Phosphodiesterase inhibitors and uses thereof

ABSTRACT

The invention provides for compounds that are phosphodiesterase inhibitors. The invention further provides for a method for screening compounds that bind to and modulate a phosphosdiesterase protein. The invention also provides methods for treating conditions associated with accumulated amyloid-beta peptide deposit accumulations by administering a phosphodiesterase-binding compound to a subject.

This application is a continuation of U.S. patent application Ser. No. 13/167,540, filed on Jun. 23, 2011, which is a continuation-in-part of International Application No. PCT/US2009/058813, filed Sep. 29, 2009, which claims priority to U.S. Provisional Application Ser. No. 61/140,315, filed Dec. 23, 2008, and International Application No. PCT/US2009/039129, filed Apr. 1, 2009, each of which are hereby incorporated by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. R21AG027468 awarded by the National Institute in Aging. The United States Government has certain rights in the invention.

All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by memory loss, synaptic dysfunction and accumulation of amyloid β-peptides (Aβ). It is caused in part by increased levels of amyloid-β-peptide 1-42 (Aβ42). Phosphodiesterase 5 (PDE5) inhibitors are widely used drugs against erectile dysfunction and pulmonary hypertension. These inhibitors are believed to increase cGMP levels which enhances phosphorylation of the transcription factor and memory-affecting molecule cAMP-responsive element binding (CREB) through activation of cGMP-dependent-protein kinases.

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), nucleotide biological second messengers, regulate various biological processes, such as blood flow regulation, cardiac muscle contraction, cell differentiation, neural transmission, glandular secretion, and gene expression. Intracellular receptors for these molecules include cyclic nucleotide phosphodiesterases (PDEs), cyclic nucleotide dependent protein kinases (PGK), and cyclic nucleotide-gated channels. PDEs are a large family of proteins that catalyze the hydrolysis of 3′,5′-cyclic nucleotides to the corresponding 5′ monophosphates. There are eleven related, but biochemically distinct, human PDE gene groups. Some PDEs are specific for hydrolysis of cAMP (such as PDE4, PDE7, and PDE8), and some are cGMP specific (such as PDE5, PDE6, and PDE9), while some PDEs have mixed specificity (such as PDE1, PDE2, PDE3, PDE10, and PDE11).

PDE 5 inhibitors are cyclic guanosine 3′,5′-monophosphate type five cGMP PDE inhibitors, which include, but are not limited to, sildenafil, tadalafil, zaprinast, and vardenafil. PDE5 inhibitors increase cGMP levels by inhibiting the degradative action of PDE5 on cGMP. No PDE inhibitor has reached the marketplace for diseases of the CNS, and no PDE5 inhibitors have been used for the treatment of Ad.

SUMMARY OF THE INVENTION

The invention is directed to a class of quinoline-containing compounds with PDE5 inhibitory potency, high selectivity, and blood-brain-barrier (BBB) permeability. In one aspect, the compound is Formula V as described herein. In one embodiment, the compound is Formula V-1 as described herein. In another embodiment, the compound is Formula V-1a as described herein. In a further embodiment, the compound is Formula V-1a1 as described herein. In some embodiments, the compound is selected from Formula V, Formula V-1, Formula V-1a, and Formula V1a1 as described herein; wherein R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷. In some embodiments, R¹ is C₃-C₈ cycloalkyl. In some embodiments, R¹ is C₃-C₈ cycloalkyl or —NR⁷R⁸. In some embodiments, R¹ is —NR⁷R⁸. In some embodiments, the compounds are those compounds depicted as compounds 1-18 as described herein. In specific embodiments, the compound is compound 3 (YF012403) or compound 8 (YF016203).

In various aspects, the invention provides a method for screening compounds to treat conditions associated with accumulated amyloid-beta peptide deposits, the method comprising: (a) selecting (or identifying or screening for) a PDE5 inhibitor compound that can modulate secretase activity for at least 1 month after completion of administration of the PDE5 inhibitor compound in an animal model of amyloid-beta peptide deposit accumulation.

In one aspect, the invention provides a method for screening compounds to treat conditions associated with accumulated amyloid-beta peptide deposits, the method comprising: (a) selecting a PDE5 inhibitor compound that comprises one or both of the following features: (i) the compound interacts with two or more amino acid residues of a phosphodiesterase protein, wherein the amino acid residues comprise F787, L804, I813, M816, or a combination thereof; or (ii) the 2nd bridging ligand (BL2) between the compound and a phosphodiesterase protein is OH—.

In one aspect, the invention provides a method for identifying a phosphodiesterase-binding compound to treat conditions associated with accumulated amyloid-beta peptide deposits, wherein the method comprises selecting a PDE5 inhibitor compound having one or more of the following features: (a) the IC₅₀ of the compound is no more than about 1000 nM; (b) the selectivity of the compound is at least a 50 fold greater potency towards PDE5 relative to PDE1, PDE2, PDE3, PDE4, PDE6, PDE7, PDE8, PDE9, PDE10, or PDE11; (c) the PDE5 inhibitory activity in vitro has an IC₅₀ no more than about 50 nM; (d) the compound penetrates the blood brain barrier; (e) the compound hydrolyzes cGMP by about 20% to about 80%; (f) the 2nd bridging ligand (BL2) between the compound and a phosphodiesterase protein is OH; or (g) the compound interacts with two or more amino acid residues of a phosphodiesterase protein, wherein the amino acid residues comprise F787, L804, I813, M816, or a combination thereof. 7. The phosphodiesterase in feature (g) can comprise, for example, phosphodiesterase type V (PDE5) or even another PDE. In one aspect, feature (g) is where the compound interacts with at least all four amino acid residues F787, L804, I813, and M816 of PDE5. In one aspect, the compound can decrease the activity or expression of a phosphodiesterase type V (PDE5) protein

In some aspects, the above described methods further comprise testing whether the selected PDE5 inhibitor can modulate secretase activity for at least 1 month after administration in an animal model of amyloid-beta peptide deposit accumulation. The secretase can be α-secretase or β-secretase. The modulation can comprise a decrease in β-secretase activity or expression levels and/or an increase in α-secretase activity or expression levels. In some aspects, the modulated secretase activity or expression of β-secretase remains decreased. In some aspects, the modulated secretase activity or expression of α-secretase remains increased. In some aspects, the modulated secretase activity persists more than 2 months, 3 months, 4 months, 5 months, 6 months, or 7 months after completion of the dosage period.

In some aspects of the above described methods, the animal model of amyloid-beta peptide deposit accumulation comprises an APP/PS1 double transgenic mouse. Where the animal model comprises this transgenic mouse, in some aspects, the step of testing whether the selected PDE5 inhibitor can modulate secretase activity for at least 1 month after administration in the APP/PS1 double transgenic mouse comprises: (a) administering the selected PDE5 inhibitor to APP/PS1 double transgenic mice for a dosage period up to about 21 days; (b) testing whether the selected PDE5 inhibitor modulates secretase activity or expression in the APP/PS1 double transgenic mice immediately after completion of the dosage period as compared to a negative control; and (c) testing whether modulated secretase activity or expression in the APP/PS1 double transgenic mice from step (b) persists more than 1 month after completion of the dosage period as compared to a negative control.

In some aspects for the above methods, the selecting step of the compound based on features can involve in silico screening, molecular docking, in vivo screening, in vitro screening, or a combination thereof.

In some aspects relating to the above methods, a dosage period of the PDE5 inhibitor compound to the animal model subject is up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 days, up to about 17 days, up to about 18 days, up to about 19 days, or up to about 20 days.

In some aspects relating to the above methods, the compound has a molecular mass less than about 500 Da, a polar surface area less than about 90 Å², less than 8 hydrogen bonds, or a combination thereof in order to penetrate the blood brain barrier.

In some aspects relating to the above methods, the PDE5 inhibitor compound has been first pre-screened by a method comprising: (a) providing an electronic library of test compounds; (b) providing atomic coordinates listed in Table 1 for at least 20 amino acid residues for the active site of the PDE5 protein, wherein the coordinates have a root mean square deviation therefrom, with respect to at least 50% of Cα atoms, of not greater than about 2 Å, in a computer readable format; (c) converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the PDE5 protein; (d) performing a data processing method, wherein electronic test compounds from the library are docked onto the three dimensional model of the PDE5 protein; and (e) determining which test compound fits into the active site of the three dimensional model of the PDE5 protein, thereby identifying which compound would bind to PDE5. In one aspect, this method can further comprise: (f) synthesizing or obtaining the compound determined to dock to the active site of the PDE5 protein; (g) contacting the PDE5 protein with the compound under a condition suitable for binding; and (h) determining whether the compound modulates PDE5 protein expression or mRNA expression, or PDE5 protein activity using a diagnostic assay.

In some aspects of the present methods, the PDE5 inhibitor compound comprises Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ie, Formula IIa, Formula IIb, Formula IIc, Formula IId, Formula IIe, Formula IIIa, Formula IIIb, Formula IIIc, Formula IIIa-1, Formula IIIb-1, Formula IIIc-1, Formula IIId, Formula IIIe, Formula IIIf; Formula IVa, Formula IVb, Formula V, Formula V-1, Formula V-1-a, or Formula V-a-1 (such as any one of compounds 1-18), as described herein. In some embodiments, the compound is selected from Formula V, Formula V-1, Formula V-1a, and Formula V1a1 as described herein; wherein R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷. In some embodiments, R¹ is C₃-C₈ cycloalkyl. In some embodiments, R¹ is C₃-C₈ cycloalkyl or —NR⁷R⁸. In some embodiments, R¹ is —NR⁷R⁸.

In some aspects, the PDE5 inhibitor decreases PDE5 protein or mRNA expression, or PDE5 activity by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, or 100%.

In some aspects, the PDE5 inhibitor has an IC50 at least about 0.1 nM, at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, or at least about 1000 nM.

In some aspects, methods for selecting a PDE5 inhibitor can comprise detecting whether the inhibitor can cause an increase or decease in a secondary messenger concentration. The secondary messenger can comprise, for example, cyclic GMP, protein kinase G (PKG), or a combination thereof. The detection can comprise an assay that measures an intracellular concentration of GTP, cyclic GMP, protein kinase G (PKG), or CREB.

In some aspects, the PDE5 inhibitor compound binds to the active site of phosphodiesterase type V (PDE5).

In some aspects, the compound has an IC50 at least about 0.1 nM, at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, or at least about 1000 nM.

In one aspect, the invention provides a method for increasing α-secretase protein activity or expression in a subject, the method comprising: (a) administering to the subject an effective amount of a composition comprising a PDE5 inhibitor compound, thereby increasing α-secretase protein activity or expression in the subject.

In one aspect, the invention provides a method for decreasing β-secretase protein activity or expression in a subject, the method comprising: (a) administering to the subject an effective amount of a composition comprising a PDE5 inhibitor compound, thereby decreasing β-secretase protein activity or expression in the subject.

In one aspect, the invention provides a method for reducing amyloid beta (Aβ) protein deposits in a subject, the method comprising: (a) administering to the subject an effective amount of a composition comprising a PDE5 inhibitor compound, thereby decreasing Aβ protein deposits in the subject.

In some aspects, the subject exhibits abnormally elevated amyloid beta plaques. In some aspects, the subject is afflicted with Alzheimer's disease, Lewy body dementia, inclusion body myositis, or cerebral amyloid angiopathy. In some aspect, the subject is a mouse, dog, cat, horse, cow, sheep, or human.

In some aspects, the compound that is administered to the subject comprises Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ie, Formula IIa, Formula IIb, Formula IIc, Formula IId, Formula IIe, Formula IIIa, Formula IIIb, Formula IIIc, Formula IIIa-1, Formula IIIb-1, Formula IIIc-1, Formula IIId, Formula IIIe, Formula IIIf; Formula IVa, Formula IVb, Formula V, Formula V-1, Formula V-1-a, or Formula V-a-1 (such as any one of compounds 1-18). In some embodiments, the compound is selected from Formula V, Formula V-1, Formula V-1a, and Formula V1a1 as described herein; wherein R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷. In some embodiments, R¹ is C₃-C₈ cycloalkyl. In some embodiments, R¹ is C₃-C₈ cycloalkyl or —NR⁷R⁸. In some embodiments, R¹ is —NR⁷R⁸. In some aspects, the compound is sildenafil, tadalafil, or vardenafil. In some aspects, the administration comprises subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; infusion; oral or nasal delivery; or a combination thereof. In some aspects the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, or at least about 10 mg/kg body weight. In other aspects, the effective amount of the administered compound is at least about 3 mg/kg body weight. In some aspects, the composition is administered at least once daily for up to 18 days, up to 19 days, up to 20 days, up to 21 days, up to 22 days, up to 23 days, up to 24 days, or up to 25 days. In some aspects, the αsecretase protein activity or expression is increased up to 3 months post-treatment, up to 4 months post-treatment, up to 5 months post-treatment, or up to 6 months post-treatment. In some aspects, the β-secretase protein activity or expression is decreased up to 3 months post-treatment, up to 4 months post-treatment, up to 5 months post-treatment, or up to 6 months post-treatment. In some aspects, the Aβ protein deposit comprises an Aβ40 isomer, an Aβ42 isomer, or a combination thereof.

In some aspects, PDE5 inhibitor compounds that are administered to subjects to modulate secretase activity or expression are administered infrequently due to the finding provided herein that PDE5 inhibitors can cause a long-lasting or sustained affect on secretase activity long-after administration. Thus, in some aspects, methods of treatment are provided where subjects are administered PDE5 inhibitors for short-term periods on a regular, but infrequent basis. For example, administration can comprise a dosage regimen comprising 1 week, 2 weeks, 3 weeks, a month, or more, followed by a period of no administration that comprises 1 week, 2 weeks, 3 weeks, a month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or more, wherein this dosage regimen can then be repeated and varied. Namely, the dosage regimen comprises a period of PDE5 inhibitor administration followed a period of no drug administration, optionally followed by further cycles. The benefit of such a cyclic regimen can be, for example, to lessen the possibility of side-effects due to total drug intake-load over time.

BRIEF DESCRIPTION OF THE FIGURES

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FIG. 1A is a graph of field input-output relationship for different stimulation intensities (5-35 V) that shows that BST is similar in 3-month-old APP/PS1 animals and WT littermates. The slope of the input-output curve at stimulation intensity equal to 35 V was ˜97% of WT littermates in APP/PS1 mice (APP/PS1 mice: 1.21±0.08 V/sec., n=7 slices from 6 males; WT mice: 1.25±0.06 V/sec., n=7 slices from 6 males). A two-way ANOVA showed no difference between double transgenic mice and their littermate controls [F_((1,12))=0.05, P=0.81]. Similar results were observed when plotting the fEPSP slope versus the amplitude of the fiber afferent volley.

FIG. 1B is a graph representing that ten minutes perfusion with sildenafil (50 nM) reverses LTP impairment in APP/PS1 mice (sildenafil-treated APP/PS1 mice equal to −100% of vehicle-treated WT littermates at 120 min. after tetanus, vs. ˜65% in vehicle-treated APP/PS1 mice; sildenafil-treated APP/PS1 mice: 215.08±11.85% at 120 min. after tetanus, n=8 slices from 7 males; vehicle-treated APP/PS1 mice: 128.47±16.79%, n=9 slices from 7 males, [F_((1,15))=4.98, P=0.041]). The inhibitor has no effect on basal neurotransmission either during its application or 120 minutes after the end of the application in experiments where no tetanic stimulation is applied (˜96% of vehicle-treated WT slices in sildenafil-treated APP/PS1 slices, vs. ˜97% in vehicle-treated transgenic slices; APP/PS1: 99.99±3.71% in sildenafil-treated slices, n=4 slices from 4 males, vs. 100.88±1.02% in vehicle-treated slices, n=5 slices from 4 males, [F_((1,7))=1.15, P=0.31]). Arrows indicate time and pattern of the tetani in this and the following figures. Bars represent the time of the application of the drug or vehicle in this and the following figures. Sildenafil reverses the impairment of LTP in the CA1 region of slices from 3-month-old APP/PS1 mice

FIG. 1C is a graph showing that sildenafil (50 nM) does not affect LTP in WT mice. Levels of LTP at 120 min. after tetanus were ˜99% of vehicle-treated WT slices (vehicle-treated WT mice: 214.28±17.49%, n=10 slices from 8 males, sildenafil-treated WT mice: 213.26±13.66%, n=9 slices from 8 males, [F_((1,17))=0.23, P=0.63]). The inhibitor has no effect on basal synaptic responses either during its application or 120 minutes after the end of the application in experiments where no tetanic stimulation is applied (˜97% of vehicle-treated WT slices; 100.51±2.34% in sildenafil-treated slices, n=5 slices from 4 males, vs. 103.72±3.86% in vehicle-treated slices, n=4 slices from 4 males, [F_((1,7))=0.26, P=0.62]). These experiments were interleaved with those of APP/PS1 mice.

FIG. 2A is a graph that shows sildenafil ameliorates cognitive dysfunction in 3-month-old APP/PS1 mice. Sildenafil (3 mg/Kg, i.p.) improves contextual fear conditioning in 3 month-old APP/PS1 mice. APP/PS1 and WT littermates treated with sildenafil or vehicle show no difference in freezing prior to training (baseline; one-way ANOVA among the four groups: F_((3,79))=2.39, P=0.07), whereas contextual FC performed 24 hrs after training shows a reduction of freezing responses in APP/PS1 mice treated with vehicle compared to vehicle-treated WT littermates [the freezing time of vehicle-treated APP/PS1 mice was ˜63% of vehicle-treated WT mice; 21.36±3.94% in APP/PS1, n=21 (12 males and 9 females), vs. 33.81±4.05% in WT littermates, n=20 (11 males and 9 females), F_((1,39))=6.64, P=0.01]. Treatment with sildenafil ameliorates deficit in freezing responses in APP/PS1 mice (the freezing time of sildenafil-treated APP/PS1 mice was about 87% of vehicle-treated WT mice: 28.95±3.94%, n=22 (12 males and 10 females), F_((1,40))=0.73, P=0.39). Sildenafil has no effect on the freezing responses of WT mice compared to vehicle-treated WT littermates [about 89% of vehicle-treated WT mice: 30.1±3.11%, n=20 (11 males and 9 females), F_((1,38))=0.52, P=0.47). Cued fear conditioning was similar among the four groups [F_((3,79))=0.89, P=0.44].

FIG. 2B is a graph demonstrating that sildenafil ameliorates cognitive dysfunction in 3-month-old APP/PS1 mice. Sildenafil (3 mg/Kg, i.p.) improves spatial working memory in 3 month-old APP/PS1 mice. APP/PS1 mice treated with vehicle do not learn the position of the hidden platform compared to vehicle-treated WT littermates [APP/PS1: 5.91±0.19 errors in the first acquisition trial (A1), 4.83±0.52 errors by the fourth consecutive trial (A4), and 5.29±0.31 errors by the recall trial (R), n=8 (4 males and females); WT: A1=5.36±0.27 errors, A4=1.63±0.39 errors, R=2.00±0.32 errors, n=10 (5 males and females)]. Two-way ANOVA showed a significant difference between the performance of vehicle-treated APP/PS1 mice and that of vehicle-treated WT littermates (F_((1,16))=39.66, P<0.0001), and planned comparisons showed that the 2 groups were significantly different at trial A2 (P=0.05), A3 (P=0.02), A4 and R (P=<0.0001). However, treatment with sildenafil ameliorates the performance of double transgenic littermates compared to vehicle-treated APP/PS1 [A1=5.87±0.22 errors, A4=3.39±0.39 errors, R=3.57±0.31 errors, n=111 (6 males and 5 females), F_((1,17))=5.99, P=0.02]. Planned comparisons showed that the 2 groups were significantly different at trial A4 (P=0.03) and R (P=0.001). Sildenafil does not affect the performance of WT mice compared to vehicle-treated WT mice [sildenafil: A1=5.5±0.23 errors, A4=1.5±0.21 errors, R=1.9±0.35 errors, n=10 (5 males and females), F_((1,18))=0.09, P=0.76).

FIG. 3A is a bar graph that shows sildenafil (3 mg/Kg, i.p. for 3 weeks at the age of 3 months) ameliorates contextual fear conditioning in transgenic mice. After 24 hours, there is a reduction of freezing behavior in APP/PS1 mice compared to WT, rescued by sildenafil treatment [˜97% of vehicle-treated WT mice in sildenafil-treated APP/PS1 mice, vs. ˜66% in vehicle-treated APP/PS1 mice; 30.32±1.98%, n=12 (7 males and 5 females) in sildenafil-treated APP/PS1 mice, 20.66±2.42%, n=17 (10 males and 7 females) in vehicle-treated APP/PS1 mice, F_((1,27))=7.10, P=0.013]. Sildenafil did not increase the freezing time in WT littermates compared to WT mice treated with vehicle [˜104% of vehicle-treated WT mice; 32.41±1.88%, n=17 (10 males and 7 females) vs. 31.16±1.21%, n=14 (8 males and 6 females), F_((1,29))=0.28, P=0.06]. There were no significant differences between the 4 groups in the cued conditioning test [F_((3,56))=0.07, P=0.097].

FIG. 3B is a graph that shows impairment of performance during radial-arm water maze testing in APP/PS1 mice is rescued by treatment with sildenafil (3 mg/Kg, i.p. for 3 weeks at the age of 3 months). There was a significant difference between the number of errors made by vehicle-treated APP/PS1 and WT littermates [APP/PS1: 6.04±0.21 errors in the first acquisition trial A1, 5.38±0.34 errors in A2, 5.14±0.36 in A3, 4.52±0.33 by the fourth consecutive trial A4, and 4.95±0.28 errors by the recall trial R, n=7 (4 males and 3 females); WT: A1=5.60±0.62 errors, A2=3.46±0.34 errors, A3=2.66±0.82 errors, A4=1.86±0.16 errors, R=2.13±0.34 errors, n=5 (3 males and 2 females)]. Two-way ANOVA revealed a significant overall difference between the performance of the two groups [F_((1,10))=33.98, P=0.0001] and planned comparison showed that the difference became pronounced since the second acquisition trial A2 (P=0.003) and in the recall trial (P=0.0001). Sildenafil improves the performance of APP/PS1 mice and does not affect the performance of WT mice [sildenafil-treated APP/PS1: A1=6.16±0.39, A2=4.16±0.53, A3=3.5±0.41, A4=2.83±0.61, R=3±0.75 errors, n=4 (3 males 1 female); sildenafil-treated WT animal: A1=6±0.29, A2=3.22±0.56, A3=2.5±0.36, A4=1.83±0.16, R=2.33±0.36 errors, n=6 (4 males and 2 females), F_((1,7))=3.42, P=0.1 and F_((1,8))=4.04, P=0.07 compared to vehicle-treated WT animals]. Statistical analysis revealed a strong effect of treatment in APP/PS1 mice treated with sildenafil compared to APP/PS1 animals treated with vehicle (F_((1,9))=18.53, P=0.002), and planned comparisons showed that the groups were significantly different at A3 (P=0.019), A4 (P=0.026) and R(P=0.017).

FIG. 3C is a graph depicting that the performance of APP/PS1 mice in the Morris water maze is improved by previous treatment with sildenafil (3 mg/Kg, i.p. for 3 weeks at the age of 3 months). Vehicle-treated transgenic mice needed 44.83±3.77 seconds to find the hidden platform after six sessions compared to 28.91±3.94 seconds required by WT littermates [˜155% of the time needed by the vehicle-treated WT mice; F_((1,21))=13.73, P=0.001; planned comparisons showed that the groups were significantly different at the fourth, fifth and sixth sessions; P=0.001, P=0.009, P=0.008, respectively]. Previous treatment with sildenafil reduces the time needed to find the platform in APP/PS1 mice (32.92±3.50 seconds, ˜114% of the time required by vehicle-treated WT mice; F_((1,16))=2.97; P=0.10). Sildenafil did not affect the performance in WT littermates that needed 25.64±3.16 seconds (˜89% of the time required by vehicle-treated WT mice; F_((1,23))=0.981; P=0.37). Statistical analysis revealed a significant difference in the overall performance of sildenafil-treated APP/PS1 mice compared to that of vehicle-treated APP/PS1 mice [F_((1,15))=0.85, P=0.02)] and planned comparisons of latency on each individual session revealed that the difference was significant at the fourth, fifth and sixth session (P=0.0001, P=0.011, P=0.034, respectively).

FIG. 3D is a bar graph demonstrating that APP/PS1 mice previously treated with sildenafil search significantly more time in the target quadrant (TQ), where the platform was located during training, than do vehicle-treated APP/PS1 littermates, during the probe test. (F_((1,16))=8.42, P=0.01). WT mice spent 33.37±2.10% of their time given in the TQ then in other quadrants [F_((3,44))=3.85, P=0.016]. Planned comparisons confirmed that they spent significantly more time in the TQ than in the adjacent quadrant to the right (AR), in the adjacent quadrant to the left (AL), or in the opposite quadrant (OQ) (TQ versus AR, P=0.03; TQ versus OQ, P=0.04; TQ versus AL, P=0.02). Sildenafil improved the performance of the APP/PS1 mice [32.25±0.58% of their time given spent in TQ, or ˜97% of the time used by vehicle-treated WT littermates, than in other quadrants; F_((3,24))=14.73; P<0.0001]. Planned comparisons confirmed that they spent significantly more time in the TQ than in the AR, in the AL, or in the OQ (P<0.0001). Vehicle-treated APP/PS1 mice spent only 21.39±1.46% of the time in the TQ [F_((3,40))=0.52, P=0.66], or ˜64% of the time used by vehicle-treated WT littermates. Sildenafil-treated WT mice spent 33.35±1.38% of the time in the TQ [F_((3,48))=3.63, P=0.019; planned comparisons TQ versus AR, P=0.03; TQ versus OQ, P=0.04; TQ versus AL, P=0.02], or ˜100% of the time used by vehicle-treated WT littermates. Statistic revealed a significant difference in the percent of time spent in the TQ by sildenafil-treated APP/PS1 mice compared with that of vehicle-treated APP/PS1 mice (F_((1,16))=8.42, P=0.01). No statistically significant difference was found in the percent of time spent in the TQ by sildenafil-treated APP/PS1 mice compared with vehicle-treated and sildenafil-treated WT mice (F_((1,17))=0.04, P=0.84; F_((1,18))=0.03, P=0.85, respectively).

FIG. 4A is a graph showing that BST impairment in 6-8 month-old APP/PS1 animals is improved by sildenafil-treatment (3 mg/Kg, i.p. for 3 weeks at the age of 3 months) [APP/PS1: ˜57% of vehicle-treated WT mice; 0.62±0.09 V/sec., (n=6 slices from 5 males), F_((1,9))=7.26, P=0.02; compared to vehicle-treated WT mice; APP/PS1+sildenafil: ˜160% of vehicle-treated APP/PS1 mice; 1.03±0.12 V/sec., (n=8 slices from 6 males), F_((1,12))=5.51, P=0.03 compared to vehicle-treated transgenic mice]. No statistically significant difference was found in the values of fEPSP slope in sildenafil-treated APP/PS1 mice compared to vehicle-treated and sildenafil-treated WT mice (F_((1,11))=0.07, P=0.79; F_((1,12))=0.03, P=0.84, respectively). Sildenafil does not change BST in WT mice [slope of the input-output curve in sildenafil-treated WT mice: ˜94% of vehicle-treated WT mice; sildenafil-treated WT mice: 1.01±0.12 V/sec. (n=6 from 5 males), vehicle-treated mice: 1.08±0.11 V/sec. (n=5 slices from 5 males); two-way ANOVA F_((1,9))=0.34, P=0.57]. Similar results were observed when plotting the fEPSP slope versus the amplitude of the fiber afferent volley.

FIG. 4B is a graph demonstrating that sildenafil (3 mg/Kg, i.p. for 3 weeks at the age of 3 months) rescues the LTP impairment in 6-8 month-old APP/PS1 mice [APP/PS1+sildenafil: ˜100% of sildenafil-treated WT mice, 233.81±30.47% of baseline at 120 min, n=7 slices from 6 males, F_((1,12))=1.18, P=0.29 compared to sildenafil treated-WT littermates; APP/PS 1+vehicle: ˜65% of vehicle-treated WT mice, 135.56±22.02% of baseline, n=7 slices from 6 males, F_((1,12))=14.82, P=0.002 compared to vehicle-treated WT littermates].

FIG. 4C is a graph depicting that sildenafil (3 mg/Kg, i.p. for 3 weeks at the age of 3 months) does not affect LTP in 6-8 month-old WT mice [˜112% of vehicle-treated mice in sildenafil-treated mice; sildenafil-treated mice: 234.67±17.19% of baseline, n=7 slices from 6 males; vehicle-treated mice: 210.01±16.62% of baseline, n=7 slices from 6 males, F_((1,12))=2.16, P=0.16]. No differences were noted in the baseline transmission of the four groups of mice in the absence of tetanus (n=4 slices from 4 animals for each group, F_((3,12))=0.17, P=0.91).

FIG. 5A are immunofluorescence photographs showing representative examples of hippocampal slices stained with a phospho-CREB antibody. The slices are fixed 60 minute after either vehicle or sildenafil (50 nM) with tetanus in 3-months old WT and APP/PS1 animals. Left, lower-power (4×) view of the entire slice. Right, higher power (16×) view of CA1 cell pyramidal area. Sildenafil re-establishes normal increase in CREB phosphorylation following tetanic stimulation in APP/PS1 mice.

FIG. 5B is a bar graph showing that the increase in the intensity of immunofluorescence (IF) in the CA1 cell body area after application of the tetanus does not appear in 3 month old APP/PS1 mice (WT: 160.51±14.96% of control, n=4; t₍₆₎=3.44, P=0.014 compared to control non-tetanized slices; APP/PS1: 111.89±6.62% of control, n=4; t₍₆₎=2.96, P=0.025 compared to tetanized slices). Sildenafil (sild) re-establishes increase in CREB phosphorylation in APP/PS1 mice after tetanus whereas it does not affect phospho-CREB levels in WT tetanized slices (APP/PS1+sildenafil: 162.58±17.09% of control, n=4; t₍₆₎=0.09, P=0.93; WT+sildenafil: 163.54±13.52% of control, n=4; t₍₆₎=0.15, P=0.88 compared to tetanized slices of WT mice).

FIG. 5C is a bar graph demonstrating that daily injections of sildenafil (3 mg/Kg, i.p. for 3 weeks in 3-month-old APP/PS1 mice) re-establish the tetanus-induced increase in CREB phosphorylation in hippocampal slices from the same mice at 6-8 months of age (WT: 151.60±8.25% of control, n=4/4; t₍₆₎=4.70, P=0.003 compared to non-tetanized slices; APP/PS1: 114.96±9.12% of control, n=4, t₍₆₎=2.96, P=0.025, compared to tetanized slices from their WT littermates; APP/PS11+sildenafil: 163.6±15.14% of control, n=4; t₍₆₎=0.69, P=0.53 compared to tetanized slices of vehicle-treated WT animals, (t₍₆₎=2.75, P=0.041 compared to tetanized slices of vehicle-treated APP/PS1 animals). Sildenafil does not affect the increase in CREB phosphorylation in WT mice (151.73±12.24% of control, n=4, t₍₆₎=0.009, P=0.993, compared to tetanized slices from WT mice).

FIG. 6A is a bar graph showing that daily injections of sildenafil for 3 weeks decreases Aβ₄₀ and Aβ₄₂ levels in 3-month-old transgenic mice (Aβ₄₀: ˜64% of vehicle-treated APP/PS1 mice, Aβ₄₂: ˜80%; Aβ₄₀ and Aβ₄₂ values were 1.71±0.22 and 3.56±0.23 ng/mg cortex, respectively, in sildenafil-treated APP/PS1 mice, n=7, versus 2.67±0.35 and 4.43±0.34 ng/mg cortex in vehicle-treated APP/PS1 mice, n=7, t₍₁₂₎=2.26, P=0.043, and t₍₁₂₎=2.09, P=0.058, respectively). Sildenafil decreased Aβ levels in 3-4 month-old APP/PS1.

FIG. 6B are photographs of immunoblots from the brains of APP/PS1 3-month-old transgenic mice treated with sildenafil (Right Column) or vehicle (Left Column) stained for APP full length, sAPPα, sAPPβ, CT83, CT99. Tubulin was used as a control.

FIG. 6C is a bar graph demonstrating that sildenafil treated 3-month-old transgenic mice do not show a change in APP levels (125.75±4.44% of control in vehicle-treated mice vs. 119.75±8.99% in vehicle-treated mice, n=4/4, t₍₆₎=0.59, P=0.572).

FIG. 6D is a bar graph demonstrating that sildenafil treated 3-month-old transgenic mice do not show a change in sAPPα (86.25±5.02% vs. 87.75±4.90, n=4/4, t₍₆₎=0.21, P=0.838).

FIG. 6E is a bar graph demonstrating that sAPPβ is decreased in 3-month old transgenic animals treated with sildenafil (43.25±3.90% vs. 62.75±4.58%, n=4/4, t₍₆₎=3.68, P=0.010). Sildenafil modifies β-secretase activity in 3-4 month-old APP/PS1.

FIG. 6F is a bar graph demonstrating that CT83 and CT99 fragments show an increase after sildenafil treatment of 3-month-old transgenics (CT83: 74.5±6.73% vs. 40.00±7.73%, n=4/4, t₍₆₎=3.36, P=0.015; CT99: 95.25±5.66% vs. 70.5±7.70%, n=4/4, t₍₆₎=2.58, P=0.041).

FIG. 7A is a bar graph showing that daily injections of sildenafil at age 3 months for 3 weeks decrease Aβ₄₀ and Aβ₄₂ levels in mice at 7-10 months of age (Aβ₄₀: ˜65% of vehicle-treated APP/PS1 mice, Aβ₄₂: ˜73%; Aβ₄₀ and Aβ₄₂ values were 49.59±7.93 and 32.55±3.27 ng/mg cortex, respectively, in sildenafil-treated APP/PS1 mice, n=6, versus 76.98±8.88 and 44.77±4.08 ng/mg in vehicle-treated APP/PS1 mice, n=7, t₍₁₁₎=2.26, P=0.045, and t₍₁₁₎=2.24, P=0.047, respectively).

FIG. 7B are photographs of immunoblots from the brains of APP/PS1 mice at 7-10 months of age that were treated with sildenafil or vehicle for 3 weeks at 3-months of age, which were stained for APP full length, sAPPα, sAPPβ, CT83, CT99.

FIG. 7C is a bar graph showing that sildenafil treated mice do not show a change in APP levels (116.33±3.32% vs. 107.4±3.88%, n=3/5, t₍₆₎=1.51, P=0.180). Daily injections of sildenafil for 3 weeks in 3-month-old APP/PS1 mice did not change APP in the same mice at 7-10 months of age.

FIG. 7D is a bar graph showing that sAPPα is increased (97±3.12% vs. 153.65±11.95, n=3/4, t₍₅₎=3.52, P=0.017) in APP/PS1 mice at 7-10 months of age that received daily injections of sildenafil for 3 weeks at 3-months of age.

FIG. 7E is a bar graph showing that sAPPβ is decreased in APP/PS1 mice at 7-10 months of age that received daily injections of sildenafil for 3 weeks at 3-months of age (28.33±2.84% vs. 25.6±2.83%, n=3/5, t₍₆₎=2.84, P=0.029).

FIG. 7F is a bar graph showing that no differences are observed for CTFs (CT83: 75.66±1.92% vs. 82.75±6.78%, n=3/5, t₍₆₎=1.13, P=0.299; CT99: 96.66±5.29% vs. 87.2±7.63%, n=3/5, t₍₆₎=0.85, P=0.427) in APP/PS1 mice at 7-10 months of age that received daily injections of sildenafil for 3 weeks at 3-months of age.

FIG. 8A is a graph demonstrating that tadalafil (50 nM) reverses LTP impairment in APP/PS1 mice (levels of LTP: tadalafil-treated APP/PS1 mice equal to ˜96% of vehicle-treated WT littermates at 120 min. after tetanus, vs. ˜56% in vehicle-treated APP/PS1 mice; tadalafil-treated APP/PS1 mice: 209.49±13.89% at 120 min. after tetanus, n=8 slices from 6 males; vehicle-treated APP/PS1 mice: 123.14±5.98%, n=8 slices from 7 males; vehicle-treated WT mice: 219.88±19.35%, n=8 slices from 7 males, F_((1,14))=15.57, P=0.001]. Tadalafil does not change basal neurotransmission either during its application or 120 minutes after the end of the application in experiments where no tetanic stimulation is applied [F_((1,6))=1.007, P=0.93]. PDE5 inhibition reverses the impairment of LTP in the CA1 region of slices from 3-month-old APP/PS1 mice.

FIG. 8B is a graph showing that ten minutes perfusion with tadalafil (50 nM) does not change the amplitude of LTP [˜106% of vehicle-treated tetanized WT slices, 232.45±21.92% vs. 219.88±19.35%, n=8/8, F_((1,14))=0.24, P=0.62) and baseline in WT mice (˜98% of vehicle-treated WT slices, 103.65±1.63% vs. 104.71±6.13, n=4/4, F_((1,6))=1.05, P=0.34]. These experiments were interleaved with those of APP/PS1 mice.

FIG. 8C is a graph demonstrating that IC354 (1 μM) does not reverse LTP impairment in APP/PS1 mice [levels of LTP: IC354-treated APP/PS1 mice equal to ˜58% of vehicle-treated WT littermates at 120 min. after tetanus, vs. ˜57% in vehicle-treated APP/PS1 mice; IC354-treated APP/PS1 mice: 129.33±8.71% at 120 min. after tetanus, n=5 slices from 5 males; vehicle-treated APP/PS1 mice: 126.81±12.39%, n=5 slices from 5 males; vehicle-treated WT mice: 220.82±9.49%, n=5 slices from 5 males, F_((1,8))=0.03, P=0.85]. IC354 does not affect basal neurotransmission either during its application or 120 minutes after the end of the application in experiments where no tetanic stimulation is applied [F_((1,6))=0.006, P=0.94].

FIG. 8D is a graph depicting a ten minutes perfusion with IC354 (1 μM) does not affect LTP in WT mice [˜102% of vehicle-treated WT slices, 226.05±18.76%, n=5 slices from 5 males, F_((1,8))=0.84, P=0.38]. The inhibitor has no effect on basal synaptic responses either during its application or 120 minutes after the end of the application in experiments where no tetanic stimulation is applied [F_((1,6))=0.072, P=0.79]. These experiments were interleaved with those of APP/PS1 mice.

FIG. 9 is a bar graph that tadalafil does not ameliorate cognition in 3-month-old APP/PS1 mice. Tadalafil (1 mg/Kg, i.p.) does not modify contextual fear conditioning in 3 month-old APP/PS1 mice. APP/PS1 and WT littermates treated with tadalafil or vehicle show no difference in freezing prior to training [F_((3,39))=0.26, P=0.853]. Fear conditioning performed 24 hrs after training shows a reduction of freezing responses in APP/PS1 mice treated with vehicle compared to vehicle-treated WT littermates [freezing time in vehicle-treated APP/PS1 mice is ˜47% of vehicle-treated WT mice; 15.34±3.15% in APP/PS1, n=12 (6 males, 6 females), vs. 33.03±5.52% in WT littermates, n=10 (5 males, 5 females), F_((1,20))=8.19, P=0.011]. Treatment with tadalafil does not rescue freezing behavior in APP/PS1 mice compared to vehicle-treated APP/PS1 animals [freezing time of tadalafil-treated APP/PS1 mice is ˜122% of vehicle-treated APP/PS1 mice: 18.76±3.89%, n=8 (4 males, 4 females), F_((1,18))=0.08, P=0.778]. Tadalafil does not affect the freezing responses of WT mice [˜85% of vehicle-treated WT mice: 28.29±3.30%, n=13 (7 males, 6 females), F_((1,21))=0.58, P=0.453]. Cued fear conditioning was similar among the four groups [F_((3,39))=0.21, P=0.884].

FIG. 10A is a graph that demonstrates that four groups of mice show no difference in the time needed to find a visible platform [APP/PS1 mice treated with sildenafil, 26.73±3.43 seconds in the first session of testing and 21.14±3.28 seconds in the fourth one; APP/PS1 mice treated with vehicle, 29.33±4.62 seconds and 20.49±4.20 seconds in the first and fourth sessions, respectively; WT mice treated with sildenafil, 26.64±3.11 seconds and 20.17±4.01 seconds in the first and fourth sessions, respectively; WT mice treated with vehicle, 26.88±2.58 seconds and 19.49±2.65 seconds in the first and fourth sessions, respectively, F_((3,35))=0.02, P=0.994]. APP/PS1 mice do not show any sensory impairment at 3 months of age.

FIG. 10B is a bar graph that shows no difference in swimming speed among the four groups was also found [APP/PS1 mice treated with sildenafil, 18.21±1.96 cm/s, APP/PS1 mice treated with vehicle, 18.65±2.31 cm/s, WT mice treated with sildenafil, 17.42±1.72 cm/s, and WT mice treated with vehicle, 18.42±1.81 cm/s, F_((3,35))=0.073; P=0.974]. APP/PS1 mice do not show any motor impairment at 3 months of age.

FIG. 11A is a graph demonstrating visible platform trials do not reveal any significant difference in the time to reach the platform among the 4 groups [APP/PS1 mice treated with sildenafil, 24.40±2.06 seconds in the first session of testing and 20.37±2.35 seconds in the fourth one; APP/PS1 mice treated with vehicle, 24.40±3.49 seconds and 21.09±2.83 seconds in the first and fourth sessions, respectively; WT mice treated with sildenafil, 22.79±2.51 seconds and 20.91±2.21 seconds in the first and fourth sessions, respectively; WT mice treated with vehicle, 21.16±3.11 seconds and 19.90±2.50 seconds in the first and fourth sessions, respectively; F_((3,39))=0.01, P=0.997]. APP/PS1 mice do not show any sensory impairment at 7-10 months of age. These animals received daily injections of sildenafil for 3 weeks at 3 months of age.

FIG. 11B is a bar graph depicting that the four groups of mice do not show any difference in swimming speed [APP/PS1 mice treated with sildenafil, 15.81±1.88 cm/s, APP/PS1 mice treated with vehicle, 17.05±1.45 cm/s, WT mice treated with sildenafil, 16.97±1.49 cm/s, and WT mice treated with vehicle, 17.36±1.34 cm/s, F_((3,39))=0.023; P=0.995]. APP/PS1 mice do not show any/motor impairment at 7-10 months of age. These animals received daily injections of sildenafil for 3 weeks at 3 months of age.

FIG. 12 is a schematic showing the fused planar ring system structures in reported PDE5 inhibitors.

FIG. 13 are chemical structures depicting four classes of structurally related, and formally independent scaffolds (I-IV) based on structure analysis of reported PDE5 inhibitors and known Structure-Activity Relationship (SAR) data.

FIG. 14 is a schematic showing the synthesis of compounds comprising scaffold Ia.

FIG. 15 is a schematic showing the synthesis of compounds comprising scaffold IIa-c.

FIG. 16 is a schematic showing the synthesis of compounds comprising scaffold IId.

FIG. 17 is a schematic showing the synthesis of compounds comprising scaffold III.

FIG. 18 is a schematic showing the synthesis of compounds comprising scaffold IVa.

FIG. 19 is a schematic of the NO/cGMP/CREB pathway.

FIG. 20 is a schematic of APP processing. Administration of the PDE5 inhibitor sildenafil modifies APP process in APP/PS1 mice. A decrease in sAPPβ levels was detected in 3-month-old APP/PS1 mice treated with sildenafil, while an increase in CT83 and CT99 fragments was observed. A persistent decrease in sAPPβ levels and a persistent increase in sAPPα levels was detected at 7-10 months of age in APP/PS1 mice that were previously treated with sildenafil when 3 months old.

FIG. 21 is a schematic of a model depicting the action of PDE5 inhibitors on synaptic plasticity, memory, and amyloid-beta (Aβ) peptide synthesis and degradation. PDE5 inhibitors can increase synaptic plasticity in APP/PS1 mice; increase memory, fear conditioning and RAWM in APP/PS1 mice; increase CREB phosphorylation in APP/PS1 mice; and can decrease Aβ peptide levels in APP/PS1 mice.

FIG. 22 shows the effect of APP and PS1 transgene overexpression onto active boutons in cell cultures. FIG. 22A are photographs of Examples of FM 1-43 staining of active release sites before and after glutamate in WT and APP/PS1 hippocampal cultures. Scale bar, 15 μm. FIG. 22B is a graph showing basal number of active boutons per unit-length-neurite was higher in cultures from Tg mice compared to WT littermates. FIG. 22C is a graph demonstrating the percent increase in presynaptic active boutons 30 min after glutamate in 0 Mg++ in WT and APP/PS1 cultures. Glutamate increased active bouton number in WT but not in APP/PS1 cultures.

FIG. 23 represents the experimental set-up. A schematic drawing of a transverse hippocampal slice is shown in the top image. Schaeffer collateral fibers and CA1 stratum radiatum are marked. Positions of the stimulating and recording electrodes are indicated. Long-term potentiation (LTP) was induced by a theta-burst stimulation of Schaeffer collateral fibers. Photograph of the interface recording chamber used for electrophysiological experiments is shown in the bottom image.

FIG. 24 represents a synthetic Scheme of new PDE5 inhibitors. Based on the requirement for new PDE5 inhibitors, a class of quinoline derivatives was designed.

FIG. 25 depicts some synthetic scheme examples. Based on the SAR, YF012403 (cyclopropyl lead) and YFO 16203 (dimethylamino lead). were picked for further investigation.

FIG. 26 represents the IC₅₀s of synthesized compounds. YF012403 and YF016203 are highlighted in red.

FIG. 27 depicts the in vitro selectivity of PDE5 inhibitors. Two compounds, YF012403 and YF016203, were picked up based on the SAR for selectivity profiling. a) Data obtained by BPS Bioscience; b) Graeme L. Card, et. al. Structure, 2004, 12, 2233-2247; c) I Saenz de Tejada, et al., International Journal of Impotence Research, 2001, 13, 282-290; d) Alain, Daugan, et. Al, Journal of Medicinal Chemistry, 2003, 46, 4533-4542.

FIG. 28 represents a pharmacokinetics profile. One compound, YF012403, was identified based on the in vitro activity and selectivity for PK profiling as compared to sildenafil.

FIG. 29 represents a pharmacokinetics profile. The graphs depicts a concentration/time curve of candidate YF012403 and sildenafil in brain tissue and plasma. The data were collected with male C57/BALB/c mice; three mice for each point.

FIG. 30 depicts a synthetic route for process chemistry of the dimethylamino derivative (YF016203).

FIG. 31 is a graph showing electrophysiology data. YF012403 reverses LTP impairment in the CA1 region of slices from 3-4 month-old mice treated with 200 nM oligomeric Aβ 42. A Two-way ANOVA was carried out: Aβ compared to Aβ plus YF012403=F(1,11)=6.073; p=0.0314.

FIG. 32 depicts a synthesis scheme of Intermediate A. Dashed lines in the scheme indicate a prophetic reaction.

FIG. 33 depicts a synthesis scheme of Intermediate B.

FIG. 34 depicts a synthesis scheme of Intermediate C.

FIG. 35 depicts a synthesis scheme of Intermediate D.

FIG. 36 depicts a synthesis scheme of Formula E.

FIG. 37 depicts a synthesis scheme of Formula F.

FIG. 38 depicts the general synthesis method of scheme A.

FIG. 39 depicts synthesis Scheme I for compound 9a (YF012403; the cyclopropyl lead).

FIG. 40 depicts synthesis Scheme II for compound 11a (YF016203; dimethylamino lead).

FIG. 41 depicts synthesis Scheme III-A1 for intermediate 10a.

FIG. 42 are graphs that show the expression levels of PDE5 mRNA in heart, whole brain, hippocampus and cerebrum of humans. In FIG. 42A, the values were normalized to β-actin mRNA. In FIG. 42B, the values shown in FIG. 42A were normalized to respective heart mRNA levels.

FIG. 43 shows the structures of cGMP-based molecules.

FIG. 44 shows the structures of β-carbolines-derived molecules.

FIG. 45 shows the structures of pyrazolopyridine, phthalazine and quinoline derivatives.

FIG. 46 shows the structures of isoquinazolinone and isoquinolinone derivatives.

FIG. 47 is a graph depicting PDE5 activity where 100 nM of cGMP substrate was used.

FIG. 48 is a Concentration-Time curve of YF012403 in mouse brain tissue and plasma (n=3 mice per group).

FIG. 49 are graphs that show the beneficial effect of YF012403 on Aβ42-induced synaptic and cognitive dysfunction. FIG. 49A shows that YF012403 ameliorates the LTP deficit in Aβ42-treated slices. The graph represents the average of the last 5 min of recording at 60 min after the tetanus. FIG. 49B shows that YF012403 ameliorates the contextual fear memory deficit in Aβ42-infused mice.

FIG. 50 is a schematic showing modifications at C8 of YF012403.

FIG. 51 is a schematic showing modifications at C3 of YF012403.

FIG. 52 is a schematic showing modifications at C3 of YF012403.

FIG. 53 is a schematic showing modifications at C3 of YF012403.

FIG. 54 is a schematic showing modifications at other positions of YF012403.

FIG. 55 are graphs that show the acute beneficial effects of sildenafil on cognitive dysfunction of 5 month-old J20 mice during contextual fear conditioning (FC) (FIG. 55A) and RAWM (FIG. 55B) testing.

FIG. 56 are graphs showing that a brief perfusion of hippocampal slices with sildenafil reverses CA1-LTP impairment in 3-month-old APP/PS1 mice. The graph in FIG. 56A shows that BST is similar in 3-month-old APP/PS1 animals and WT littermates. Summary graph of EPSP slopes versus fiber volley amplitudes for different stimulation intensities ranging from 5 to 35 V [35 V: ˜97% of WT littermates in APP/PS1 mice, n=7 slices from 6 males vs. n=7 slices from 6 males in WT slices; two-way ANOVA: F_((1,12))=0.05, P=0.81]. There is not difference in fiber volley between WT and transgenic animals (F_((1,12))=3.97, P=0.06). FIG. 56B is a dose-response curve that shows the effect of different concentrations of sildenafil on synaptic plasticity in slices from transgenic animals. The minimum effective dose that completely rescues synaptic plasticity is 50 nM (n=6 slices from 6 males for each group). FIG. 56C is a graph showing that sildenafil (50 nM) ameliorates LTP in slices from APP/PS1 mice that were potentiated through 1 or 2 series of theta-burst stimulations (1 tetanus: t_((1,10))=3.38, P=0.007 compared to vehicle-treated APP/PS1 slices; 2 tetani: t_((1,10))=3.92, P=0.003; 3 tetani: t_((1,10))=13.47, P<0.001; n=6 slices from 6 males for each group). Slices from WT mice that received one theta-burst stimulation showed a significant increase in LTP when they were perfused with 50 nM sildenafil compared to vehicle-treated WT slices (1 tetanus: t_((1,10))=2.25, P=0.048; 2 tetani: t_((1,10))=1.37, P=0.200; 3 tetani: t_((1,10))=1.26, P=0.236; n=6 slices from 6 males for each group).

FIG. 57 are graphs that show three month old APP/PS1 mice have normal BST associated with no changes in AMPA- and NMDA-receptor currents. FIGS. 57A-B show normalized current-voltage plots of AMPA receptor (AMPAR) (FIG. 57A) and NMDA receptor (NMDAR) (FIG. 57B) currents from adult WT (n=3 cells) and APP/PS1 (n=3 cells) CA1 pyramidal cells. AMPAR-mediated EPSCs were normalized to the EPSC at −90 mV. NMDAR-mediated EPSCs were normalized to the NMDA response at +50 mV. FIG. 57C is a comparison of AMPAR to NMDAR current ratio in the WT and APP/PS1 pyramidal cells. The ratio was calculated by dividing the amplitude of the AMPAR current measured at −70 mV by the NMDAR current measured 50 ms after the peak at +50 mV.

FIG. 58 are graphs showing that sildenafil ameliorates cognitive function in 3-month-old APP/PS1 mice. FIG. 58A shows that the minimum concentration of sildenafil needed to improve contextual fear memory in APP/PS1 mice is 3 mg/kg. A concentration of 1.5 mg/kg does not improve freezing, whereas 6 mg/kg has the same effect as 3 mg/kg [1.5 mg/kg sildenafil: n=8 (4 males, 4 females) vs. vehicle treated animals n=14 (7 males, 7 females), F_((1,20))=0.82, P=0.375; 3 mg/kg sildenafil: n=8 (4 males, 4 females), F_((1,20))=11.58, P=0.003; 6 mg/kg sildenafil: n=8 (4 males, 4 females), F_((1,20))=8.48, P=0.009. FIG. 58B shows that the minimum concentration of sildenafil needed to improve spatial working memory in APP/PS1 mice is 3 mg/kg for 3 weeks. A concentration of 1.5 mg/kg does not improve RAWM performance, whereas 6 mg/kg has the same effect as 3 mg/kg [1.5 mg/kg sildenafil: n=8 (4 males, 4 females) vs. vehicle treated animals n=14 (7 males, 7 females), F_((1,20))=0.82, P=0.375 and F_((1,20))=0.05, P=0.824 for A4 and R, respectively; 3 mg/kg sildenafil: n=8 (4 males, 4 females), F_((1,20))=11.58, P=0.003 and F_((1,20))=11.36, P=0.003; 6 mg/kg sildenafil: n=8 (4 males, 4 females), F_((1,20))=8.48, P=0.009 and F_((1,20))=7.12, P=0.015]. FIG. 58C is a summary graph showing that the minimum time needed for sildenafil to have a positive effect on spatial working memory in APP/PS1 mice is 2 weeks with a concentration of 3 mg/kg [1 week: F_((1,20))=1.81, P=0.19 and F_((1,20))=0.82, P=0.386 for A4 and R, respectively; 2 weeks: F_((1,20))=9.69, P=0.005 and F_((1,20))=10.35, P=0.004; 3 weeks: F_((1,20))=13.19, P=0.002 and F_((1,20))=11.36, P=0.003; n=8 (4 males, 4 females) for each condition].

FIG. 59 is a graph that shows that sildenafil does not modify cued conditioning in 3 months old mice. Vehicle-treated APP/PS1 mice have similar performance as vehicle-treated WT littermates (F_((1,39))=0.16, P=0.691). Injections of sildenafil (3 mg/kg) do not affect freezing during cued conditioning in APP/PS1 mice and WT littermates (F_((1,38))=1.2, P=0.279 and F_((1,40))=0.08, P=0.773 compared to vehicle-treated WT mice, respectively).

FIG. 60 is a graph that shows rescue by sildenafil of contextual fear memory impairment in APP/PS1 mice is complete with a high intensity foot shock eliciting high amounts of freezing [F(1,26)=52.24; P=0.001].

FIG. 61 are graphs showing that tadalafil does not ameliorate cognition in 3-month-old APP/PS1 mice. FIG. 61A shows that tadalafil (1 mg/Kg, i.p.) does not modify contextual fear conditioning in 3 month-old APP/PS1 mice. APP/PS1 and WT littermates treated with tadalafil or vehicle show no difference in freezing prior to training [F(3,39)=0.26, P=0.853]. Fear conditioning performed 24 hrs after training shows a reduction of freezing responses in APP/PS1 mice treated with vehicle compared to vehicle-treated WT littermates [freezing time in vehicle-treated APP/PS1 mice is ˜47% of vehicle-treated WT mice; n=12 (6 males, 6 females), vs. n=10 (5 males, 5 females) for WT littermates, F(1,20)=8.19, P=0.011]. Treatment with tadalafil does not rescue freezing behavior in APP/PS1 mice compared to vehicle-treated APP/PS1 animals [freezing time of tadalafil-treated APP/PS1 mice is ˜122% of vehicle-treated APP/PS1 mice: n=8 (4 males, 4 females), F(1,18)=0.08, P=0.778]. Tadalafil does not affect the freezing responses of WT mice [˜85% of vehicle-treated WT mice: n=13 (7 males, 6 females), F(1,21)=0.58, P=0.453]. FIG. 61B shows that cued fear conditioning is similar among the four groups [F(3,34)=1.42, P=0.253]. FIG. 61C shows that tadalafil does not improve spatial working memory in 3 month-old APP/PS1 mice. APP/PS1 mice treated with tadalafil do not learn the position of the hidden platform compared to vehicle-treated APP/PS1 [APP/PS1+tadalafil: n=8 (4 males and females); APP/PS1+vehicle: n=8 (5 males and females); F(1,14)=0.71, P=0.736 and F(1,14)=2.46, P=0.139 for A4 and R, respectively]. Tadalafil does not affect the performance of WT mice compared to vehicle-treated WT mice [WT+tadalafil: n=8 (4 males and females); WT+vehicle: n=8 (4 males and females); F(1,14)=0.32, P=0.579 and F(1,14)=0.09, P=0.763 for A4 and R, respectively].

FIGS. 62A-H show graphs that demonstrate the minimum concentration and duration of treatment with sildenafil needed in 3-month-old APP/PS1 mice to improve both associative and spatial memory in 6- to 8-month-old APP/PS1 mice. FIG. 62A is a summary graph showing that the minimum concentration of sildenafil needed to improve contextual fear memory is 3 mg/kg [n=8 (4 males, 4 females) for each condition in this and the following panels; F(1,14)=6.5, P=0.023 at 3 mg/kg sildenafil]. FIG. 62B is a summary graph showing that the minimum time needed for sildenafil to have a positive effect on contextual fear memory is 2 weeks with a concentration of 3 mg/kg [F(1,14)=13.9, P=0.002 at 2 weeks]. FIG. 62C is a summary graph showing that the minimum concentration of sildenafil needed to improve spatial working memory is 3 mg/kg [A4: F(1,14)=12.7, P=0.001 and R: F(1,14)=13.6, P=0.002 at 3 mg/kg sildenafil]. FIG. 62D is a summary graph showing that the minimum time needed for sildenafil to have a positive effect on spatial working memory is 2 weeks with a concentration of 3 mg/kg [A4: F(1,14)=12.9, P=0.001 and R: F(1,14)=9.6, P=0.008 at 2 weeks]. FIG. 62E is a summary graph showing that the minimum concentration of sildenafil needed to improve the performance with the MWM is 3 mg/kg (the graph shows the time needed to reach the platform in the last trial of the hidden platform) [F(1,14)=16.9, P=0.001 with 3 mg/kg sildenafil]. FIG. 62F is a summary graph showing that the minimum concentration of sildenafil needed to improve the performance with the probe trial is 3 mg/kg (the graph shows the percentage of time spent in the target quadrant—TQ) [F(1,14)=18.3, P=0.001 at 3 mg/kg sildenafil]. FIG. 62G is a summary graph showing that the minimum time needed for sildenafil to improve the performance with the MWM is 2 weeks with a concentration of 3 mg/kg [F(1,14)=16.8, P=0.001 at 2 weeks]. FIG. 62H is a summary graph showing that the minimum time needed for sildenafil to improve the performance with the probe trial is 2 weeks with a concentration of 3 mg/kg [F(1,14)=19.8, P=0.001 at 2 weeks].

FIG. 63 shows graphs that demonstrate the minimum concentration and duration of treatment with sildenafil needed in 3-month-old APP/PS1 mice to improve BST and LTP as they reach 6- to 8-months of age. FIG. 63A is a summary graph showing that the minimum concentration of sildenafil needed to improve BST is 3 mg/kg [n=8 males for each condition in this and following panels; F(1,14)=23.32, P<0.001 at 3 mg/kg sildenafil]. FIG. 63B is a summary graph showing that the minimum concentration of sildenafil needed to improve LTP is 3 mg/kg [F(1,14)=70.3, P<0.001 at 3 mg/kg sildenafil]. FIG. 63C is a summary graph showing that the minimum time needed for sildenafil to have a positive effect on BST is 2 weeks with a concentration of 3 mg/kg [F(1,14)=39.4, P<0.001 at 2 weeks]. FIG. 63D is a summary graph showing that the minimum time needed for sildenafil to have a positive effect on LTP is 2 weeks with a concentration of 3 mg/kg [F(1,14)=64.5, P<0.001 at 2 weeks].

FIG. 64 is a dose-response curve showing the effect of different concentrations of sildenafil, applied for 10 min through the bath solution, on BST and LTP in slices from 6 month old APP/PS1 animals. FIG. 64A is a graph that shows different concentrations of sildenafil do not change fEPSP slope (F(4,30)=0.09, P=0.985). FIG. 64B is a graph that shows the minimum effective dose of inhibitor that completely rescues LTP is 500 nM (93% of vehicle-treated WT slices; t(1,11)=7.04, P<0.001, n=7/4 for various groups).

FIGS. 65A-D are graphs showing that sildenafil decreases Aβ levels in APP/PS1 mice. FIG. 65A shows that sildenafil decreases Aβ₄₀ and Aβ₄₂ levels in 3-month-old transgenic mice with a minimum effective dose of 3 mg/kg (t₍₁₂₎=2.32, P=0.039 and t₍₁₂₎=2.30, P=0.04 for Aβ₄₀ and Aβ₄₂, respectively; n=7 for various groups). FIG. 65B shows that daily injections of sildenafil for 3 weeks in 3-month-old APP/PS1 mice reduce Aβ levels in the same mice at 7-10 months of age. The minimum effective dose that decreases Aβ₄₀ and Aβ₄₂ levels is 3 mg/kg (t₍₁₂₎=2.22, P=0.04 and t₍₁₂₎=2.85, P=0.01 for Aβ₄₀ and Aβ₄₂, respectively; n=7 for various groups). FIG. 65C shows that the minimum time needed for 3 mg/kg sildenafil to have a positive effect on Aβ₄₀ is 2 weeks (t₍₁₂₎=2.43, P=0.03) whereas values of Aβ₄₂ levels did not reach significance at this time (2 weeks: t₍₁₂₎=2.22, P=0.04; 3 weeks: t₍₁₂₎=2.85, P=0.01; n=7 for various groups). FIG. 65D shows that the minimum time needed for 3 mg/kg sildenafil, administered at 3 months of age, to have a positive effect on Aβ₄₀ levels in the same mice at 7-8 months of age is 3 weeks (t₍₁₂₎=2.33, P=0.03) whereas levels of Aβ₄₂ at 2 weeks are slightly above significance (2 weeks: t₍₁₂₎=2.02, P=0.06; 3 weeks: t₍₁₂₎=2.95, P=0.01; n=7 for various groups).

FIG. 66 shows that YF012403 (10 min prior to tetanus) rescues the defect in LTP in slices from transgenic mice.

FIG. 67 and FIG. 68 show the effects of YF012403 in a transgenic mouse model of Alzheimer's disease. The graphs show that behavioral defects for 2 day radial arm water maze, as well as contextual fear memory are attenuated by treatment with the inhibitor. This is a transgenic mouse model of amyloid beta elevation.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for a class of quinoline-containing compounds which have excellent PDE5 inhibitory potency, high selectivity, reasonable pharmacokinetics and good permeability across the blood-brain-barrier (BBB). These compounds may be used to minimize the side effects for AD patients, the third most costly disease in the U.S. The compounds of the invention may also be used to treat erectile dysfunction (ED), pulmonary hypertension, cardiovascular disorder, diabetes, and GI disorders.

In some embodiments, the invention provides methods for identifying PDE5 inhibitors that can cause a sustained or long-term decrease in β-secretase activity or expression in a subject. In one embodiment, the invention provides methods that select for PDE5 inhibitors that can cause a decrease in β-secretase activity or expression in a subject well after administration of the PDE5 inhibitor has ended. For example, PDE5 inhibitors can be screened or selected based on their ability to cause a decrease in β-secretase activity or expression in an animal model of Aβ accumulation (such as APP/PS1 mice) for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more.

To shrink the candidate pool of PDE5 inhibitor compounds to be tested in Aβ accumulation animal models, PDE5 inhibitors can first be screened or selected based on their possession of certain characteristics, such as having one or more of: an IC₅₀ no greater than about 100 nM; a selectivity that is at least 50-fold greater for PDE5 than for other PDEs; a PDE5 inhibitory activity in vitro that has an IC₅₀ no greater than about 50 nM, the ability to penetrate the BBB; the ability to hydrolyze cGMP by at least about 20% (or at least about 80%); an interaction between the compound and PDE5 that comprises a second bridging ligand that is a hydroxyl group; and an interaction between the compound and PDE5 that comprises contacts with PDE5 at amino acid residues F787, L804, I813, M816, or a combination thereof (including contacts at all four residues).

In some embodiments, the candidate pool of PDE5 inhibitors to be tested in Aβ accumulation animal models can first be screened or selected based on “medicinal chemistry” strategies described herein (see Examples). For example, based on the structure analysis of reported PDE5 inhibitors and known SAR data (FIG. 12, four class of structurally related, but nevertheless formally independent scaffolds I-IV (see FIG. 13), are deemed as PDE5 inhibitor candidates. Compounds derived from these scaffolds can first be screened and optimized on computational models. Compounds with highest score will be synthesized and tested for potency. At this stage, the synthetic effort will be guided by the testing results of potency/selectivity. Compounds with satisfactory potency and selectivity (lead compounds) will be further studied for PK, bioavailability/brain penetration and off-target activities (safety). Selected compounds can be tested in the Aβ accumulation animal models to determine whether they cause a sustained a sustained or long-term decrease in β-secretase activity or expression. As used herein, a PDE5 inhibitor compound does not necessarily preclude the possibility that the compound may also be able to inhibit other PDEs.

Thus, the disclosure provides for the discovery that PDE5 inhibitor compounds display a prolonged and protective effect against synaptic dysfunction and memory loss that persists beyond the administration of the inhibitor. In some embodiments, PDE5 inhibitor compounds are desired and screened or selected for that have a prolonged inhibitory affect on β-secretase while having a prolonged enhancing effect on α-secretase. In some embodiments, methods of screening for therapeutic agents (for conditions associated with amyloid-β-peptide accumulation, such as AD) involve testing whether an agent exerts a prolonged inhibitory affect on β-secretase activity or expression and/or a prolonged stimulatory affect α-secretase activity or expression.

In some embodiments, the invention is directed at identifying and using agents that interact with Aβ targets that lead to neuronal dysfunction. The invention also provides for compounds that modulate PDE5 protein expression or activity, or that modulate activity or expression of secretases (for example, α- and β-secretase). For example, the compounds can be PDE5 inhibitors, a class of compounds that counteract the progression of neurodegenerative diseases, such as AD (Puzzo et al [12]). Currently used AD therapies (such as acetylcholinesterase inhibitors or NMDA antagonists) have limited efficacy.

Alzheimer's Disease

Alzheimer's disease (AD) is characterized by neuronal loss, extracellular senile plaques and intracellular neurofibrillary tangles, leading to memory loss. AD purportedly begins as a synaptic disorder produced at least in part, by Aβ (Selkoe, D. J. Alzheimer's disease is a synaptic failure. Science (New York, N.Y. 298, 789-791 (2002)). Aβ-induced reduction in long-term-potentiation (LTP), a physiological correlate of synaptic plasticity that is thought to underlie learning and memory, and phosphorylation of the memory transcription factor CREB, are ameliorated by nitric oxide (NO) donors and cGMP-analogs (Puzzo, D., et al. Amyloid-beta peptide inhibits activation of the nitric oxide/cGMP/cAMP-responsive element-binding protein pathway during hippocampal synaptic plasticity. J Neurosci 25, 6887-6897 (2005)). Vice-versa, genetic ablation of NO-synthase 2 (NOS2) results in worsening of the AD phenotype in mice expressing mutated amyloid precursor protein (APP) (Colton, C. A., et al. NO synthase 2 (NOS2) deletion promotes multiple pathologies in a mouse model of Alzheimer's disease. Proceedings of the National Academy of Sciences of the United States of America 103, 12867-12872 (2006)). Taken together, these findings show that up-regulation of the NO pathway can be protective in AD.

AD is characterized neuropathologically by neuronal loss, extracellular senile plaques (SPs) and intracellular neurofibrillary tangles (NFTs). SPs are chiefly comprised of AP3 aggregates. The major component of NFTs is the microtubule binding protein tau. Clinically, AD is characterized by cognitive dysfunction and begins as a synaptic disorder that involves progressively larger areas of the brain over time [1]. An emerging view of the processes involved in synaptic impairment shows that the subtlety and variability of the earliest amnesic symptoms, occurring in the absence of any other clinical signs of brain injury, can be due to discrete changes in the function of a single synapse, produced at least in part, by Aβ [5, 7, 10, 11].

One of the important targets for developing a causal therapy for Alzheimer's disease is represented by synapses. Synaptic alterations are highly correlated with the severity of clinical dementia [1, 2], whereas other important variables such as senile plaques and neurofibrillary tangles are involved to a lesser extent [1]. The importance of synaptic alterations in AD has been confirmed by studies of transgenic (Tg) mouse models of AD [3] as well as of long-term potentiation (LTP), a widely studied cellular model of learning and memory (L&M) [4], which is impaired following application of amyloid-β (Aβ) both in slices and in vivo [3, 5-12]. Aβ has been found to markedly inhibit LTP. Electrophysiological studies using Tg, human Aβ producing mice have often revealed significant deficits in basal synaptic transmission and/or LTP in the hippocampus [23-30].

NO is a central molecule in cellular biochemical processes. The gas has been established as an important messenger molecule in various steps of brain physiology, from development to synaptic plasticity and learning and memory. In AD research, NO has been found to have a protective effect on Aβ-induced damage of the nervous system [38-40]. Studies performed on PC12 cells, sympathetic neurons and hippocampal neurons, have shown that treatment with the NO generator S-nitroso penicillamine exerts a neuroprotective effect due to the inhibition of the pro-apoptotic factor caspase-2 by nitrosylation [39], whereas inhibition of NO synthesis by N-nitro-L-arginine methyl ester does not protect against Aβ-induced neurotoxicity. Aβ has been found to impair NO generation by decreasing NMDA receptor signal transduction [38], by subtracting NADPH availability to NO-synthase (NOS) [41], or by inhibiting the phosphorylation of the serine-threonine kinase Akt [42]. Moreover, i-NOS deletion enhances AD pathology in the APP mice [43]. Thus, drugs enhancing the NO-cascade have a beneficial effect against AD [44].

Despite the neuroprotective function of NO is clear and indisputable, the gas has also been viewed as a major agent of neuropathology and cell death when it is produced in high quantity. High amounts of NO lead to generation of significant quantity of peroxinitrites that are responsible for oxidative and nitrosative stress in Aβ-induced cell death [45-51]. In fact, release of low amounts of NO by the constitutive forms of NOS that include both the neuronal and the endothelial isoforms, n-NOS and e-NOS, promotes synaptic plasticity and learning, whereas uncontrolled production of high amounts of the gas by the inducible form of NOS (i-NOS) can promote oxidative and nitrosative stress via production of peroxinitrite [45-51]. Thus, both Aβ-induced downregulation of the NO cascade which blocks plasticity and memory and generation of peroxinitrites leading to cell death, can play roles in AD. The current status of drug research exploiting these discoveries is focused both on finding ways to upregulate the NO cascade and therefore elicit neuroprotection, as well as on finding ways to block peroxinitrite toxic effects in order to limit neuropathology [52].

PDE5 Inhibition

Herein, therapeutic strategies can bypass NO production by focusing on steps at the downstream level of NO generation. PDE5, the enzyme that degrades cGMP, is such a downstream target of the disclosure's therapies aimed at treating Aβ deposits in subjects in need thereof. PDE5 is part of a superfamily of enzymes including 11 types/families of PDE (PDE1 to PDE11), some of which play a critical role in memory and behavior in diverse organisms ranging from the fruit fly, Drosophila melanogaster, to humans [53]. PDEs are multi-domain proteins, wherein about 270 amino acids localized towards the C-terminus is highly conserved between the 11 families. This domain contains the PDEs' catalytic function. Non-homologous amino acid segments have regulatory function or confer specific binding properties. PDE2, PDE5, PDE6 and PDE10 contain putative GAF domains within their regulatory amino terminal portion, which have been shown to bind cGMP.

PDE5, a cGMP specific PDE, is found in varying concentrations in various tissues such as vascular and visceral smooth muscle, platelets, and skeletal muscle. The cGMP-specific PDE is ubiquitously expressed, and can be found in several brain regions associated with cognitive function, including the hippocampus, cortex and cerebellum [17,18]. PDE5 is comprised of the conserved C-terminal, zinc containing, catalytic domain, and an N-terminal regulatory domain. The C-terminus of PDE5 catalyses the cleavage of cGMP, while the N terminus contains two GAF domain repeats, which each contains a cGMP-binding site (one of high affinity and the other of lower affinity). Regulation of PDE5 activity occurs through binding of cGMP to the high and low affinity cGMP binding sites, subsequently followed by phosphorylation, which occurs only when both sites are occupied. Inhibition of PDE5 decreases cGMP breakdown, thus allows for maintenance of cGMP levels. Sildenafil, for example, is a potent inhibitor of PDE5 and is the active ingredient of Viagra™.

Some clinically useful drugs have been developed as family-selective inhibitors of PDEs. However, none have been shown to exert long-lasting inhibitory effects on β-secretase expression or activity, as well as long-lasting excitatory effects on α-secretase expression or activity. Preclinical studies have shown that the selective PDE5 inhibitors sildenafil and vardenafil raise hippocampal cGMP levels and improve memory in aged rats (Prickaerts et al, 2002) and mice (Baratti & Boccia, 2001). In human studies sildenafil was found to enhance selective retention and verbal recognition memory in humans (Schultheiss et al, 2001). Because sildenafil (Viagra by Pfizer, pyrazol-[4,3-d]-pyrimidinone derivative) is reported to cross the blood brain barrier (BBB), it represents a good candidate for CNS studies. But evidence for vardenafil is indirect (Prickaerts, J., et al. Neurochem Int 45, 915-928 (2004)), and tadalafil is unlikely to cross it. Sildenafil has an IC₅₀ against PDE5 of 6.0 nM and an in vivo half-life of 0.4 hrs in rodents (˜4 hrs in humans) (Walker, D. K., et al. Xenobiotica 29, 297-310 (1999); Daugan, A., et al. J Med Chem 46, 4533-4542 (2003)). In addition, it is very selective for PDE5 over all of the other PDE iso-enzymes, including PDE1, which is expressed in myocardium and blood vessels besides the brain and can result in vasodilatation and tachycardia (selectivity ratio 180) (Daugan, A., et al. J Med Chem 46, 4533-4542 (2003)), and PDE6, which is expressed only in retina and can transiently disturb vision (selectivity ratio 12) (Daugan, A., et al. J Med Chem 46, 4533-4542 (2003)).

A variety of physiological processes in the nervous, cardiovascular, and immune systems are controlled by the NO/cGMP signaling pathway. For example, in smooth muscle, NO and natriuretic peptides regulate vascular tone by stimulating relaxation through cGMP. Degradation of cGMP is controlled by cyclic nucleotide PDEs, and PDE5 is the most highly expressed PDE that hydrolyzes cGMP in these cells. One effective way to up-regulate the NO pathway is by increasing cGMP levels through inhibitors of phosphodiesterase 5 (PDE5), a member of a superfamily of enzymes including 11 types of PDE, some of which play a critical role in memory and behavior in diverse organisms ranging from the fruit fly, Drosophila melanogaster to humans (Davis, 1996; Barad et al, 1998; Zhang et al, 2004). These drugs are widely used to treat erectile dysfunction and pulmonary hypertension. Thus, their side effects are known and have not precluded their use in humans. Interestingly, PDE5 is expressed in several brain regions associated with cognitive function, such as the hippocampus, cortex and cerebellum (van Staveren, W. C., Steinbusch, H. W., Markerink-van Ittersum, M., Behrends, S. & de Vente, J. Eur J Neurosci 19, 2155-2168 (2004); Van Staveren, W. C., et al. J Comp Neurol 467, 566-580 (2003)).

Cyclic GMP, which phosphorylates the transcription factor CREB and activates cGMP dependent protein kinases (PKGs) has been implicated in the modulation of neurotransmission, LTP and memory [13-16]. Elevation of the cGMP levels through the inhibition of the cGMP-degrading enzyme phosphodiesterase-5 (PDE5), an enzyme expressed in several brain regions associated with cognitive function such as the hippocampus and cortex [17,18], improves memory in aged rats [14] and mice [16]. Elevation of cGMP through the PDE5 inhibitor sildenafil (Viagra) also enhances selective retention and verbal recognition memory in humans [19]. The effects of cGMP on L&M are mediated by intra and extracellular nitric oxide (NO), a molecule whose production is stimulated by soluble guanylyl cyclase (sGC) [20-22]. Preclinical studies have shown that the selective PDE5 inhibitors sildenafil and vardenafil raise hippocampal cGMP levels and improve memory in aged rats (Prickaerts, J., de Vente, J., Honig, W., Steinbusch, H. W. & Blokland, A. Eur J Pharmacol 436, 83-87 (2002)) and mice (Baratti, C. M. & Boccia, M. M. Behav Pharmacol 10, 731-737 (1999)). Further studies using Tg Aβ-producing mice have revealed an age-dependent decrease in the phosphorylation of CREB protein, these studies have provided a clue as to the mechanisms underlying the Aβ-mediated changes in LTP [31-33]. CREB phosphorylation is required for memory formation and is regulated by cAMP levels and activated cAMP-dependent-protein kinase (PKA) [34-36] as well as by cGMP levels and activated cGMP-dependent-protein kinase (PKG) [37]. Importantly, in vitro studies report that Aβ inactivates PKA and PKG, thereby reducing cAMP, phospho-CREB and LTP [10, 12, 33]. These observations show that agents that enhance the CREB-signaling pathway and act through the NO-activated cascade have potential for the treatment of AD.

PDE5 Inhibitors optimized for CNS diseases

None of the commercially available PDE5 inhibitors were developed to have the characteristics required for administration in a chronic disease of the CNS, such as AD. Thus, in some embodiments, the invention provides methods for identifying an agent or compound for the treatment of AD (or other Aβ-accumulation related conditions) that comprise selecting the agent or compound on the basis of having one or more characteristics that make the compound optimized for treating CNS diseases. For example, the characteristics can comprise: an IC₅₀ no greater than about 100 nM; a selectivity that is at least 50-fold greater for PDE5 than for other PDEs; a PDE5 inhibitory activity in vitro that has an IC₅₀ no greater than about 50 nM, the ability to penetrate the BBB; the ability to hydrolyze cGMP by at least about 20% (or at least about 80%); an interaction between the compound and PDE5 that comprises a second bridging ligand that is a hydroxyl group; and an interaction between the compound and PDE5 that comprises contacts with PDE5 at amino acid residues F787, L804, I813, M816, or a combination thereof.

In some embodiments, the invention provides methods for identifying or designing agents or compounds for the treatment of conditions associated with Aβ accumulation, where computer aided-medicinal chemistry methods are used to identify and/or design agents or compounds tailored to satisfy one or more of the characteristics mentioned above and/or to suit the strengths of various bioassays described herein.

In some embodiments, the invention provides for PDE5 inhibitor compounds based on four scaffold structures identified through a thorough analysis of Structure-Activity Relationship (SAR) characteristics of existing PDE5 inhibitors. The scaffold structures served and will continue to serve as leads for development of future compounds [See EXAMPLE 3]. Compounds based on the four scaffold structures can be screened for having one or more of the characteristics described in paragraph [0091] above, and/or for having the ability to cause a prolonged or sustained decrease in β-secretase activity or expression in an animal model of Aβ accumulation (such as the APP/PS1 mouse).

The invention provides methods for identifying compounds which can be used for treating subjects that exhibit abnormally elevated amyloid beta plaques. In addition, the invention provides methods for identifying compounds which can be used for the treatment of Alzheimer's disease, Lewy body dementia, inclusion body myositis, or cerebral amyloid angiopathy, hypertension, and erectile dysfunction. The methods can comprise the identification of test compounds or agents (e.g., peptides (such as antibodies or fragments thereof), small molecules, nucleic acids (such as siRNA or antisense RNA), or other agents) that can bind to a PDE5 polypeptide molecule and/or have an inhibitory effect on the biological activity of PDE5 or its expression, and subsequently determining whether these compounds can modulate secretase activity and/or decrease Aβ deposits. In one embodiment, the compound is a PDE5 inhibitor.

The term “modulate”, as it appears herein, refers to a change in the activity or expression of a protein molecule. For example, modulation can cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of a secretase protein molecule.

In one embodiment, a PDE5 inhibitor compound can be a peptide fragment of a PDE5 protein that binds to the phosphodiesterase protein. For example, the PDE5 molecule can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 1 or SEQ ID NO: 2. The fragment can comprise at least about 10 amino acids, a least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, a least about 50 amino acids, at least about 60 amino acids, or at least about 75 amino acids of SEQ ID NO: 1 or SEQ ID NO: 2.

SEQ ID NO: 1 is the human wild type amino acid sequence corresponding to the PDE5 enzyme (residues 1-875; Genbank Accession No. AAI26234):

MERAGPSFGQQRQQQQPQQQKQQQRDQDSVEAWLDDHWDFTFSYFVRKAT REMVNAWFAERVHTIPVCKEGIRGHTESCSCPLQQSPRADNSAPGTPTRK ISASEFDRPLRPIVVKDSEGTVSFLSDSEKKEQMPLTPPRFDHDEGDQCS RLLELVKDISSHLDVTALCHKIFLHIHGLISADRYSLFLVCEDSSNDKFL ISRLFDVAEGSTLEEVSNNCIRLEWNKGIVGHVAALGEPLNIKDAYEDPR FNAEVDQITGYKTQSILCMPIKNHREEVVGVAQAINKKSGNGGTFTEKDE KDFAAYLAFCGIVLHNAQLYETSLLENKRNQVLLDLASLIFEEQQSLEVI LKKIAATIISFMQVQKCTIFIVDEDCSDSFSSVFHMECEELEKSSDTLTR EHDANKINYMYAQYVKNTMEPLNIPDVSKDKRFPWTTENTGNVNQQCIRS LLCTPIKNGKKNKVIGVCQLVNKMEENTGKVKPFNRNDEQFLEAFVIFCG LGIQNTQMYEAVERAMAKQMVTLEVLSYHASAAEEETRELQSLAAAVVPS AQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKHEVLCRWIL SVKKNYRKNVAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAA LSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQI LSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPH QKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIE PTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNR QKWQALAEQQEKMLINGESGQAKRN

SEQ ID NO: 2 is the mouse wild type amino acid sequence corresponding to the PDE5 enzyme (residues 1-865; Genbank Accession No. NP_700471):

MERAGPNSVRSQQQRDPDWVEAWLDDHRDFTFSYFIRKATRDMVNAWFSE RVHNIPVCKEGIRAHTESCSCSLQQSPHADNTTPGAPARKISASEFDRPL RPIVVKDSEGTVSFLSDSGKKEQMPLTPPRFDSDEGDQCSRLLELVKDIS SHLDVTALCHKIFLHIHGLISADRYTLFLVCEDSSKDKFLISRLFDVAEG STLEEASNNCIRLEWNKGIVGHVAAFGEPLNIKDAYEDPRFNAEVDQITG YKTQSILCMPIKNHREEVVGVAQAINKKSGNGGTFTEKDEKDFAAYLAFC GIVLHNAQLYETSLLENKRNQVLLDLASLIFEEQQSLEVILKKIAATIIS FMQVQKCTIFIVDEDCPDSFSRVFHMECEEVGKPSDPLTREQDANKINYM YAQYVKNTMEPLNIPDVTKDKRFPWTNENMGHVNTPCIGSLLCTPIKNGK KNKVIGVCQLVNKMEENTGKIKAFNQNDEQFLEAFVIFCGLGIQNTQMYE AVERAMAKQMVTLEVLSYHASAAEEETRELQALSAAVVPSAQTLKITDFS FSDFELSDLETALCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNV AYHNWRHAFNTAQCMFAALKAGKIQNKLTDLETLALLIAALSHDLDHRGV NNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIDEYK TTLKIIKQAILATDLALYIKRRGEFFELIRKNQFSFEDPLQKELFLAMLM TACDLSAITKPWPIQQRIAELVAAEFFDQGDRERKELNMEPADLMNREKK NKIPSMQVGFIDAICLQLYEALTHVSEDCLPLLDGCRKNRQKWQALAEQQ EKMLLNGESSQGKRD

Fragments include all possible amino acid lengths between and including about 8 and 100 about amino acids, for example, lengths between about 10 and 100 amino acids, between about 15 and 100 amino acids, between about 20 and 100 amino acids, between about 35 and 100 amino acids, between about 40 and 100 amino acids, between about 50 and 100 amino acids, between about 70 and 100 amino acids, between about 75 and 100 amino acids, or between about 80 and 100 amino acids. These peptide fragments can be obtained commercially or synthesized via liquid phase or solid phase synthesis methods (Atherton et al., (1989) Solid Phase Peptide Synthesis: a Practical Approach. IRL Press, Oxford, England). The PDE5 peptide fragments can be isolated from a natural source, genetically engineered, or chemically prepared. These methods are well known in the art.

A PDE5 inhibitor compound can also be a protein, such as an antibody (monoclonal, polyclonal, humanized, and the like), or a binding fragment thereof, directed against the phosphodiesterase enzyme, PDE5. An antibody fragment can be a form of an antibody other than the full-length form and includes portions or components that exist within full-length antibodies, in addition to antibody fragments that have been engineered. Antibody fragments can include, but are not limited to, single chain Fv (scFv), diabodies, Fv, and (Fab′)₂, triabodies, Fc, Fab, CDR1, CDR2, CDR3, combinations of CDR's, variable regions, tetrabodies, bifunctional hybrid antibodies, framework regions, constant regions, and the like (see, Maynard et al., (2000) Ann. Rev. Biomed. Eng. 2:339-76; Hudson (1998) Curr. Opin. Biotechnol. 9:395-402). Antibodies can be obtained commercially, custom generated, or synthesized against an antigen of interest according to methods established in the art (Janeway et al., (2001) Immunobiology, 5th ed., Garland Publishing).

Inhibition of RNA encoding a PDE5 protein can effectively modulate the expression of the PDE5 gene from which the RNA is transcribed. Inhibitors are selected from the group comprising: siRNA, interfering RNA or RNAi; dsRNA; RNA Polymerase III transcribed DNAs; ribozymes; and antisense nucleic acid, which can be RNA, DNA, or artificial nucleic acid.

Antisense oligonucleotides, including antisense DNA, RNA, and DNA/RNA molecules, act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the DNA sequence encoding a PDE5 polypeptide can be synthesized, e.g., by conventional phosphodiester techniques (Dallas et al., (2006) Med. Sci. Monit. 12 (4):RA67-74; Kalota et al., (2006) Handb. Exp. Pharmacol. 173:173-96; Lutzelburger et al., (2006) Handb. Exp. Pharmacol. 173:243-59).

siRNA comprises a double stranded structure containing from about 15 to about 50 base pairs, for example from about 21 to about 25 base pairs, and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. Antisense nucleotide sequences include, but are not limited to: morpholinos, 2′-O-methyl polynucleotides, DNA, RNA and the like. RNA polymerase III transcribed DNAs contain promoters, such as the U6 promoter. These DNAs can be transcribed to produce small hairpin RNAs in the cell that can function as siRNA or linear RNAs that can function as antisense RNA. The PDE5 inhibitor compound can contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited. In addition, these forms of nucleic acid can be single, double, triple, or quadruple stranded. (see for example Bass (2001) Nature, 411, 428 429; Elbashir et al., (2001) Nature, 411, 494 498; and PCT Publication Nos. WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO 01/29058, WO 99/07409, WO 00/44914).

In some embodiments, a PDE5 inhibitor can be a small molecule that binds to a phosphodiesterase protein (for example a PDE5 protein) and disrupts its function. Small molecules are a diverse group of synthetic and natural substances generally having low molecular weights. They can be isolated from natural sources (for example, plants, fungi, microbes and the like), are obtained commercially and/or available as libraries or collections, or synthesized. Candidate small molecules that inhibit PDE5 can be identified via in silico screening or high-through-put (HTP) screening of combinatorial libraries. Most conventional pharmaceuticals, such as aspirin, penicillin, and many chemotherapeutics, are small molecules, can be obtained commercially, can be chemically synthesized, or can be obtained from random or combinatorial libraries as described below (Werner et al., (2006) Brief Funct. Genomic Proteomic 5 (1):32-6).

Knowledge of the primary sequence of a molecule of interest, such as a PDE5 polypeptide, and the similarity of that sequence with other proteins of the same PDE family (such as PDE1, PDE2, PDE3, PDE4, PDE6, PDE7, PDE8, PDE9, PDE10, or PDE11), can provide information as to the inhibitors or antagonists of the protein of interest. Identification and screening antagonists can be further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of antagonists, in addition to protein agonists.

The invention provides methods for screening and identifying compounds used to treat conditions associated with accumulated amyloid-beta peptide deposits, such AD. In one embodiment, the method comprises selecting a PDE5 inhibitor compound that can modulate secretase activity for at least 1 month after completion of administration of the PDE5 inhibitor compound in an animal model of amyloid-beta peptide deposit accumulation. In another embodiment, the method comprises selecting a PDE5 inhibitor compound that comprises one or both of the following features: (a) the compound interacts with two or more amino acid residues of a phosphodiesterase protein, wherein the amino acid residues comprise F787, L804, I813, M816, or a combination thereof; or (b) the 2^(nd) bridging ligand (BL2) between the compound and a phosphodiesterase protein is OH—. In another embodiment, the method can comprise selecting a PDE5 inhibitor compound having one or more of the following features: (a) the IC₅₀ of the compound is no more than about 1000 nM; (b) the selectivity of the compound is at least a 50 fold greater potency towards PDE5 relative to PDE1, PDE2, PDE3, PDE4, PDE6, PDE7, PDE8, PDE9, PDE10, or PDE11; (c) the PDE5 inhibitory activity in vitro has an IC₅₀ no more than about 50 nM; (d) the compound penetrates the blood brain barrier; (e) the compound hydrolyzes cGMP by about 20% to about 80%; (f) the 2^(nd) bridging ligand (BL2) between the compound and a phosphodiesterase protein is OH—; or (g) the compound interacts with two or more amino acid residues of a phosphodiesterase protein, wherein the amino acid residues comprise F787, L804, I813, M816, or a combination thereof. In a further embodiment, the compound, for example the PDE5 inhibitor, has an IC₅₀ of at least about 0.1 nM, at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, or at least about 900 nM. In another embodiment, PDE5 inhibitory activity in vitro has an IC₅₀ of at least about 0.1 nM, at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, of at least about 45 nM, but no more than about 50 nM. In some embodiments, the PDE5 inhibitor compound can have a molecular mass less than about 500 Da in order to penetrate the blood brain barrier. In other embodiments, the PDE5 inhibitor compound can have a polar surface area less than about 90 Å² and should have 8 or fewer hydrogen bonds in order to penetrate the blood brain barrier. The screening and identifying of the compound can comprise in silico screening, molecular docking, in vivo screening, in vitro screening, or a combination thereof.

Test compounds, such as PDE5 inhibitor compounds, can be screened from large libraries of synthetic or natural compounds (see Wang et al., (2007) Curr Med Chem, 14 (2):133-55; Mannhold (2006) Curr Top Med Chem, 6 (10):1031-47; and Hensen (2006) Curr Med Chem 13(4):361-76). Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., (1996) Tib Tech 14:60).

Methods for preparing libraries of molecules are well known in the art and many libraries are commercially available. Libraries of interest in the invention include peptide libraries, randomized oligonucleotide libraries, synthetic organic combinatorial libraries, and the like. Degenerate peptide libraries can be readily prepared in solution, in immobilized form as bacterial flagella peptide display libraries or as phage display libraries. Peptide ligands can be selected from combinatorial libraries of peptides containing at least one amino acid. Libraries can be synthesized of peptoids and non-peptide synthetic moieties. Such libraries can further be synthesized which contain non-peptide synthetic moieties, which are less subject to enzymatic degradation compared to their naturally-occurring counterparts. Libraries are also meant to include for example but are not limited to peptide-on-plasmid libraries, polysome libraries, aptamer libraries, synthetic peptide libraries, synthetic small molecule libraries, neurotransmitter libraries, and chemical libraries. The libraries can also comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the functional groups described herein.

Small molecule combinatorial libraries can also be generated and screened. A combinatorial library of small organic compounds is a collection of closely related analogs that differ from each other in one or more points of diversity and are synthesized by organic techniques using multi-step processes. Combinatorial libraries include a vast number of small organic compounds. One type of combinatorial library is prepared by means of parallel synthesis methods to produce a compound array. A compound array can be a collection of compounds identifiable by their spatial addresses in Cartesian coordinates and arranged such that each compound has a common molecular core and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, with each compound identified and tracked by its spatial address. Examples of parallel synthesis mixtures and parallel synthesis methods are provided in U.S. Ser. No. 08/177,497, filed Jan. 5, 1994 and its corresponding PCT published patent application WO95/18972, published Jul. 13, 1995 and U.S. Pat. No. 5,712,171 granted Jan. 27, 1998 and its corresponding PCT published patent application WO96/22529, which are hereby incorporated by reference.

Examples of chemically synthesized libraries are described in Fodor et al., (1991) Science 251:767-773; Houghten et al., (1991) Nature 354:84-86; Lam et al., (1991) Nature 354:82-84; Medynski, (1994) BioTechnology 12:709-710; Gallop et al., (1994) J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., (1993) Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., (1994) Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., (1992) Biotechniques 13:412; Jayawickreme et al., (1994) Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., (1993) Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242, dated Oct. 14, 1993; and Brenner et al., (1992) Proc. Natl. Acad. Sci. USA 89:5381-383.

Examples of phage display libraries are described in Scott et al., (1990) Science 249:386-390; Devlin et al., (1990) Science, 249:404-406; Christian, et al., (1992) J. Mol. Biol. 227:711-718; Lenstra, (1992) J. Immunol. Meth. 152:149-157; Kay et al., (1993) Gene 128:59-65; and PCT Publication No. WO 94/18318.

In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058; and Mattheakis et al., (1994) Proc. Natl. Acad. Sci. USA 91:9022-9026.

In one non-limiting example, non-peptide libraries, such as a benzodiazepine library (see e.g., Bunin et al., (1994) Proc. Natl. Acad. Sci. USA 91:4708-4712), can be screened. Peptoid libraries, such as that described by Simon et al., (1992) Proc. Natl. Acad. Sci. USA 89:9367-9371, can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994), Proc. Natl. Acad. Sci. USA 91:11138-11142.

The three dimensional geometric structure of an active site, for example that of a PDE5 polypeptide can be determined by known methods in the art, such as X-ray crystallography, which can determine a complete molecular structure. Solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures. The geometric structures can be measured with a complexed ligand, natural or artificial, which can increase the accuracy of the active site structure determined. In one embodiment, a compound that binds to a PDE5 protein can be identified via: (1) providing an electronic library of test compounds; (2) providing atomic coordinates listed in Table 1 for at least 20 amino acid residues for the active site of PDE5 (see PDB Entry No. 1RKP), wherein the coordinates have a root mean square deviation therefrom, with respect to at least 50% of Cα atoms, of not greater than about 2 Å, in a computer readable format; (3) converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the PDE5 protein; (4) performing a data processing method, wherein electronic test compounds from the library are docked onto the three dimensional model of the PDE5 protein; and determining which test compound fits into the active site of the three dimensional model of the PDE5 protein, thereby identifying which compound would bind to PDE5. In another embodiment, the method can further comprise: synthesizing or obtaining the compound determined to dock to the active site of the PDE5 protein; contacting the PDE5 protein with the compound under a condition suitable for binding; and determining whether the compound modulates PDE5 protein expression or mRNA expression, or PDE5 protein activity using a diagnostic assay.

TABLE 1 Atomic Coordinates for Residues of a Phosphodiesterase Type V Crystal (see http://www.rcsb.org/pdb/explore/explore.do?structureId=1RKP). Table 1 discloses SEQ ID NOS: 9 and 10, respectively. CRYST1   74.456   74.456  130.132  90.00  90.00  120.00  P 31 2 1    6 ATOM 1 N GLU A 535 62.379 7.919 74.219 1.00 64.07 N ATOM 2 CA GLU A 535 61.655 7.334 75.386 1.00 64.13 C ATOM 3 C GLU A 535 60.151 7.585 75.248 1.00 63.42 C ATOM 4 O GLU A 535 59.719 8.722 75.040 1.00 63.16 O ATOM 5 CB GLU A 535 62.164 7.961 76.686 1.00 64.66 C ATOM 6 CG GLU A 535 61.856 7.150 77.938 1.00 65.75 C ATOM 7 CD GLU A 535 63.045 6.329 78.407 1.00 66.70 C ATOM 8 OE1 GLU A 535 64.082 6.932 78.763 1.00 66.73 O ATOM 9 OE2 GLU A 535 62.945 5.083 78.421 1.00 67.31 O ATOM 10 N GLU A 536 59.361 6.521 75.369 1.00 62.64 N ATOM 11 CA GLU A 536 57.907 6.617 75.248 1.00 61.52 C ATOM 12 C GLU A 536 57.196 6.915 76.564 1.00 60.16 C ATOM 13 O GLU A 536 56.375 7.833 76.636 1.00 59.78 O ATOM 14 CB GLU A 536 57.339 5.329 74.643 1.00 62.35 C ATOM 15 CG GLU A 536 57.611 5.170 73.154 1.00 63.55 C ATOM 16 CD GLU A 536 57.015 3.896 72.584 1.00 64.28 C ATOM 17 OE1 GLU A 536 55.799 3.669 72.770 1.00 64.70 O ATOM 18 OE2 GLU A 536 57.761 3.124 71.946 1.00 64.83 O ATOM 19 N THR A 537 57.499 6.139 77.601 1.00 58.22 N ATOM 20 CA THR A 537 56.868 6.353 78.895 1.00 56.36 C ATOM 21 C THR A 537 57.251 7.740 79.400 1.00 54.93 C ATOM 22 O THR A 537 56.556 8.322 80.227 1.00 55.10 O ATOM 23 CB THR A 537 57.303 5.291 79.924 1.00 56.64 C ATOM 24 OG1 THR A 537 56.388 5.291 81.027 1.00 56.74 O ATOM 25 CG2 THR A 537 58.704 5.584 80.436 1.00 56.58 C ATOM 26 N ARG A 538 58.360 8.268 78.892 1.00 53.09 N ATOM 27 CA ARG A 538 58.813 9.594 79.287 1.00 51.19 C ATOM 28 C ARG A 538 57.880 10.610 78.643 1.00 48.94 C ATOM 29 O ARG A 538 57.497 11.602 79.264 1.00 48.50 O ATOM 30 CB ARG A 538 60.247 9.836 78.816 1.00 53.02 C ATOM 31 CG ARG A 538 60.906 11.044 79.458 1.00 55.21 C ATOM 32 CD ARG A 538 62.339 11.222 78.983 1.00 57.48 C ATOM 33 NE ARG A 538 63.018 12.282 79.722 1.00 59.89 N ATOM 34 CZ ARG A 538 64.205 12.785 79.395 1.00 60.96 C ATOM 35 NH1 ARG A 538 64.853 12.324 78.332 1.00 61.46 N ATOM 36 NH2 ARG A 538 64.743 13.750 80.131 1.00 61.65 N ATOM 37 N GLU A 539 57.518 10.357 77.388 1.00 45.81 N ATOM 38 CA GLU A 539 56.609 11.242 76.679 1.00 42.68 C ATOM 39 C GLU A 539 55.251 11.195 77.377 1.00 40.37 C ATOM 40 O GLU A 539 54.585 12.214 77.519 1.00 39.22 O ATOM 41 CB GLU A 539 56.460 10.808 75.215 1.00 43.02 C ATOM 42 CG GLU A 539 55.432 11.626 74.449 1.00 43.19 C ATOM 43 CD GLU A 539 55.298 11.213 72.995 1.00 44.20 C ATOM 44 OE1 GLU A 539 55.160 10.000 72.723 1.00 45.03 O ATOM 45 OE2 GLU A 539 55.317 12.106 72.125 1.00 43.70 O ATOM 46 N LEU A 540 54.849 10.005 77.811 1.00 38.56 N ATOM 47 CA LEU A 540 53.576 9.835 78.503 1.00 37.86 C ATOM 48 C LEU A 540 53.597 10.591 79.829 1.00 37.30 C ATOM 49 O LEU A 540 52.619 11.232 80.210 1.00 36.88 O ATOM 50 CB LEU A 540 53.310 8.351 78.766 1.00 37.38 C ATOM 51 CG LEU A 540 52.109 8.025 79.659 1.00 37.39 C ATOM 52 CD1 LEU A 540 50.830 8.560 79.034 1.00 36.69 C ATOM 53 CD2 LEU A 540 52.023 6.516 79.857 1.00 37.22 C ATOM 54 N GLN A 541 54.720 10.498 80.532 1.00 36.78 N ATOM 55 CA GLN A 541 54.882 11.179 81.807 1.00 36.28 C ATOM 56 C GLN A 541 54.701 12.681 81.603 1.00 35.16 C ATOM 57 O GLN A 541 53.961 13.335 82.343 1.00 34.46 O ATOM 58 CB GLN A 541 56.271 10.886 82.382 1.00 37.39 C ATOM 59 CG GLN A 541 56.571 11.610 83.685 1.00 39.02 C ATOM 60 CD GLN A 541 57.923 11.232 84.260 1.00 40.18 C ATOM 61 OE1 GLN A 541 58.950 11.337 83.585 1.00 41.02 O ATOM 62 NE2 GLN A 541 57.930 10.790 85.512 1.00 40.34 N ATOM 63 N SER A 542 55.363 13.216 80.579 1.00 33.84 N ATOM 64 CA SER A 542 55.282 14.643 80.269 1.00 32.54 C ATOM 65 C SER A 542 53.883 15.091 79.858 1.00 31.82 C ATOM 66 O SER A 542 53.407 16.139 80.299 1.00 31.56 O ATOM 67 CB SER A 542 56.265 14.991 79.151 1.00 33.33 C ATOM 68 OG SER A 542 57.577 14.582 79.496 1.00 35.00 O ATOM 69 N LEU A 543 53.225 14.310 79.006 1.00 30.20 N ATOM 70 CA LEU A 543 51.887 14.674 78.558 1.00 29.51 C ATOM 71 C LEU A 543 50.901 14.617 79.721 1.00 29.02 C ATOM 72 O LEU A 543 50.123 15.542 79.924 1.00 28.83 O ATOM 73 CB LEU A 543 51.423 13.738 77.435 1.00 29.76 C ATOM 74 CG LEU A 543 49.958 13.851 76.997 1.00 28.65 C ATOM 75 CD1 LEU A 543 49.697 15.223 76.399 1.00 28.64 C ATOM 76 CD2 LEU A 543 49.644 12.751 75.989 1.00 29.19 C ATOM 77 N ALA A 544 50.946 13.530 80.489 1.00 28.94 N ATOM 78 CA ALA A 544 50.036 13.351 81.617 1.00 29.97 C ATOM 79 C ALA A 544 50.184 14.418 82.704 1.00 30.89 C ATOM 80 O ALA A 544 49.209 14.770 83.366 1.00 30.59 O ATOM 81 CB ALA A 544 50.225 11.956 82.225 1.00 29.71 C ATOM 82 N ALA A 545 51.395 14.935 82.879 1.00 31.85 N ATOM 83 CA ALA A 545 51.657 15.949 83.898 1.00 33.51 C ATOM 84 C ALA A 545 51.448 17.378 83.401 1.00 35.05 C ATOM 85 O ALA A 545 51.354 18.315 84.199 1.00 36.04 O ATOM 86 CB ALA A 545 53.076 15.795 84.416 1.00 33.74 C ATOM 87 N ALA A 546 51.374 17.549 82.086 1.00 35.09 N ATOM 88 CA ALA A 546 51.201 18.874 81.515 1.00 35.61 C ATOM 89 C ALA A 546 49.813 19.438 81.763 1.00 35.63 C ATOM 90 O ALA A 546 48.836 18.707 81.877 1.00 35.97 O ATOM 91 CB ALA A 546 51.487 18.838 80.015 1.00 36.50 C ATOM 92 N VAL A 547 49.733 20.756 81.847 1.00 35.36 N ATOM 93 CA VAL A 547 48.461 21.416 82.059 1.00 35.33 C ATOM 94 C VAL A 547 47.841 21.577 80.665 1.00 34.74 C ATOM 95 O VAL A 547 48.558 21.821 79.694 1.00 33.97 O ATOM 96 CB VAL A 547 48.679 22.785 82.745 1.00 36.26 C ATOM 97 CG1 VAL A 547 49.003 23.844 81.721 1.00 36.87 C ATOM 98 CG2 VAL A 547 47.470 23.147 83.570 1.00 36.92 C ATOM 99 N VAL A 548 46.523 21.414 80.568 1.00 33.98 N ATOM 100 CA VAL A 548 45.818 21.511 79.285 1.00 33.32 C ATOM 101 C VAL A 548 45.177 22.876 79.070 1.00 33.04 C ATOM 102 O VAL A 548 44.125 23.169 79.621 1.00 33.68 O ATOM 103 CB VAL A 548 44.700 20.438 79.174 1.00 32.34 C ATOM 104 CG1 VAL A 548 44.086 20.460 77.776 1.00 31.44 C ATOM 105 CG2 VAL A 548 45.261 19.058 79.494 1.00 32.61 C ATOM 106 N PRO A 549 45.795 23.724 78.243 1.00 33.67 N ATOM 107 CA PRO A 549 45.221 25.051 78.002 1.00 33.42 C ATOM 108 C PRO A 549 43.829 25.004 77.368 1.00 33.26 C ATOM 109 O PRO A 549 43.436 24.002 76.759 1.00 33.22 O ATOM 110 CB PRO A 549 46.274 25.728 77.126 1.00 33.75 C ATOM 111 CG PRO A 549 46.905 24.588 76.413 1.00 34.40 C ATOM 112 CD PRO A 549 47.031 23.529 77.471 1.00 33.46 C ATOM 113 N SER A 550 43.083 26.090 77.537 1.00 32.37 N ATOM 114 CA SER A 550 41.721 26.199 77.023 1.00 32.42 C ATOM 115 C SER A 550 41.629 26.072 75.510 1.00 32.15 C ATOM 116 O SER A 550 42.619 26.223 74.802 1.00 32.01 O ATOM 117 CB SER A 550 41.116 27.541 77.421 1.00 32.10 C ATOM 118 OG SER A 550 41.756 28.591 76.718 1.00 33.62 O ATOM 119 N ALA A 551 40.418 25.801 75.032 1.00 32.30 N ATOM 120 CA ALA A 551 40.150 25.667 73.608 1.00 32.75 C ATOM 121 C ALA A 551 40.486 26.982 72.903 1.00 33.76 C ATOM 122 O ALA A 551 41.063 26.986 71.809 1.00 32.78 O ATOM 123 CB ALA A 551 38.688 25.323 73.389 1.00 31.08 C ATOM 124 N GLN A 552 40.112 28.093 73.533 1.00 35.12 N ATOM 125 CA GLN A 552 40.373 29.419 72.977 1.00 36.28 C ATOM 126 C GLN A 552 41.872 29.640 72.816 1.00 35.40 C ATOM 127 O GLN A 552 42.327 30.095 71.772 1.00 35.68 O ATOM 128 CB GLN A 552 39.787 30.508 73.887 1.00 38.28 C ATOM 129 CG GLN A 552 40.175 31.927 73.474 1.00 41.97 C ATOM 130 CD GLN A 552 39.584 32.998 74.381 1.00 44.31 C ATOM 131 OE1 GLN A 552 39.738 32.952 75.604 1.00 45.94 O ATOM 132 NE2 GLN A 552 38.912 33.977 73.780 1.00 45.25 N ATOM 133 N THR A 553 42.634 29.313 73.854 1.00 35.24 N ATOM 134 CA THR A 553 44.084 29.479 73.815 1.00 35.13 C ATOM 135 C THR A 553 44.719 28.630 72.709 1.00 34.63 C ATOM 136 O THR A 553 45.675 29.057 72.059 1.00 34.04 O ATOM 137 CB THR A 553 44.727 29.081 75.166 1.00 35.91 C ATOM 138 OG1 THR A 553 44.289 29.978 76.193 1.00 36.78 O ATOM 139 CG2 THR A 553 46.246 29.119 75.073 1.00 36.40 C ATOM 140 N LEU A 554 44.177 27.432 72.497 1.00 33.49 N ATOM 141 CA LEU A 554 44.707 26.510 71.492 1.00 32.20 C ATOM 142 C LEU A 554 44.226 26.770 70.066 1.00 31.50 C ATOM 143 O LEU A 554 44.740 26.175 69.114 1.00 30.62 O ATOM 144 CB LEU A 554 44.380 25.071 71.901 1.00 31.79 C ATOM 145 CG LEU A 554 44.996 24.657 73.238 1.00 32.19 C ATOM 146 CD1 LEU A 554 44.540 23.259 73.623 1.00 31.66 C ATOM 147 CD2 LEU A 554 46.521 24.722 73.124 1.00 31.58 C ATOM 148 N LYS A 555 43.243 27.657 69.925 1.00 30.33 N ATOM 149 CA LYS A 555 42.692 28.018 68.623 1.00 30.16 C ATOM 150 C LYS A 555 41.999 26.865 67.903 1.00 29.96 C ATOM 151 O LYS A 555 41.728 26.970 66.703 1.00 29.11 O ATOM 152 CB LYS A 555 43.801 28.544 67.708 1.00 31.51 C ATOM 153 CG LYS A 555 44.678 29.622 68.306 1.00 32.51 C ATOM 154 CD LYS A 555 45.948 29.766 67.480 1.00 34.45 C ATOM 155 CE LYS A 555 47.010 30.567 68.213 1.00 35.84 C ATOM 156 NZ LYS A 555 48.314 30.485 67.485 1.00 37.56 N ATOM 157 N ILE A 556 41.695 25.781 68.615 1.00 28.78 N ATOM 158 CA ILE A 556 41.076 24.624 67.971 1.00 28.91 C ATOM 159 C ILE A 556 39.622 24.779 67.543 1.00 29.19 C ATOM 160 O ILE A 556 39.063 23.879 66.917 1.00 29.06 O ATOM 161 CB ILE A 556 41.202 23.345 68.841 1.00 28.80 C ATOM 162 CG1 ILE A 556 40.496 23.536 70.183 1.00 28.62 C ATOM 163 CG2 ILE A 556 42.671 23.010 69.044 1.00 27.87 C ATOM 164 CD1 ILE A 556 40.373 22.258 70.988 1.00 28.38 C ATOM 165 N THR A 557 39.009 25.912 67.866 1.00 29.50 N ATOM 166 CA THR A 557 37.628 26.149 67.466 1.00 30.59 C ATOM 167 C THR A 557 37.590 26.846 66.102 1.00 30.62 C ATOM 168 O THR A 557 36.543 26.918 65.464 1.00 30.24 O ATOM 169 CB THR A 557 36.883 27.031 68.495 1.00 32.28 C ATOM 170 OG1 THR A 557 37.071 26.494 69.811 1.00 32.94 O ATOM 171 CG2 THR A 557 35.387 27.063 68.186 1.00 32.13 C ATOM 172 N ASP A 558 38.745 27.341 65.656 1.00 30.86 N ATOM 173 CA ASP A 558 38.852 28.040 64.374 1.00 30.19 C ATOM 174 C ASP A 558 38.948 27.094 63.171 1.00 29.95 C ATOM 175 O ASP A 558 39.790 26.195 63.144 1.00 29.77 O ATOM 176 CB ASP A 558 40.098 28.942 64.337 1.00 31.32 C ATOM 177 CG ASP A 558 40.227 29.844 65.553 1.00 33.65 C ATOM 178 OD1 ASP A 558 39.214 30.102 66.247 1.00 35.70 O ATOM 179 OD2 ASP A 558 41.358 30.315 65.803 1.00 33.50 O ATOM 180 N PHE A 559 38.108 27.309 62.164 1.00 28.55 N ATOM 181 CA PHE A 559 38.176 26.478 60.974 1.00 28.88 C ATOM 182 C PHE A 559 39.515 26.708 60.270 1.00 28.34 C ATOM 183 O PHE A 559 40.006 25.839 59.553 1.00 27.56 O ATOM 184 CB PHE A 559 37.028 26.800 60.012 1.00 29.35 C ATOM 185 CG PHE A 559 35.697 26.246 60.443 1.00 30.27 C ATOM 186 CD1 PHE A 559 34.686 27.091 60.889 1.00 31.00 C ATOM 187 CD2 PHE A 559 35.453 24.874 60.392 1.00 30.91 C ATOM 188 CE1 PHE A 559 33.443 26.579 61.282 1.00 31.39 C ATOM 189 CE2 PHE A 559 34.223 24.349 60.779 1.00 30.09 C ATOM 190 CZ PHE A 559 33.212 25.206 61.226 1.00 31.43 C ATOM 191 N SER A 560 40.111 27.876 60.497 1.00 27.63 N ATOM 192 CA SER A 560 41.388 28.226 59.877 1.00 27.38 C ATOM 193 C SER A 560 42.605 27.663 60.605 1.00 26.39 C ATOM 194 O SER A 560 43.742 27.952 60.233 1.00 26.83 O ATOM 195 CB SER A 560 41.525 29.751 59.779 1.00 28.09 C ATOM 196 OG SER A 560 40.526 30.291 58.929 1.00 31.03 O ATOM 197 N PHE A 561 42.361 26.860 61.635 1.00 24.77 N ATOM 198 CA PHE A 561 43.424 26.255 62.430 1.00 23.76 C ATOM 199 C PHE A 561 44.554 25.643 61.607 1.00 23.00 C ATOM 200 O PHE A 561 44.324 25.035 60.561 1.00 21.56 O ATOM 201 CB PHE A 561 42.826 25.174 63.338 1.00 24.56 C ATOM 202 CG PHE A 561 43.837 24.450 64.180 1.00 24.58 C ATOM 203 CD1 PHE A 561 44.259 24.980 65.401 1.00 24.44 C ATOM 204 CD2 PHE A 561 44.343 23.215 63.772 1.00 25.19 C ATOM 205 CE1 PHE A 561 45.165 24.288 66.207 1.00 24.36 C ATOM 206 CE2 PHE A 561 45.251 22.512 64.571 1.00 25.59 C ATOM 207 CZ PHE A 561 45.663 23.049 65.794 1.00 25.48 C ATOM 208 N SER A 562 45.774 25.796 62.111 1.00 22.34 N ATOM 209 CA SER A 562 46.965 25.250 61.475 1.00 22.82 C ATOM 210 C SER A 562 47.840 24.661 62.574 1.00 22.93 C ATOM 211 O SER A 562 47.844 25.169 63.699 1.00 23.69 O ATOM 212 CB SER A 562 47.739 26.344 60.737 1.00 22.99 C ATOM 213 OG SER A 562 48.934 25.814 60.195 1.00 23.71 O ATOM 214 N ASP A 563 48.596 23.614 62.249 1.00 22.12 N ATOM 215 CA ASP A 563 49.447 22.940 63.233 1.00 22.41 C ATOM 216 C ASP A 563 50.950 23.082 62.994 1.00 22.50 C ATOM 217 O ASP A 563 51.749 22.540 63.761 1.00 21.60 O ATOM 218 CB ASP A 563 49.139 21.445 63.238 1.00 22.28 C ATOM 219 CG ASP A 563 49.639 20.761 61.982 1.00 22.77 C ATOM 220 OD1 ASP A 563 49.156 21.113 60.884 1.00 22.58 O ATOM 221 OD2 ASP A 563 50.524 19.884 62.087 1.00 24.62 O ATOM 222 N PHE A 564 51.338 23.780 61.935 1.00 22.69 N ATOM 223 CA PHE A 564 52.758 23.931 61.614 1.00 24.24 C ATOM 224 C PHE A 564 53.661 24.395 62.754 1.00 24.20 C ATOM 225 O PHE A 564 54.798 23.938 62.867 1.00 24.07 O ATOM 226 CB PHE A 564 52.949 24.876 60.428 1.00 23.91 C ATOM 227 CG PHE A 564 52.302 24.403 59.164 1.00 25.67 C ATOM 228 CD1 PHE A 564 52.254 23.044 58.852 1.00 25.98 C ATOM 229 CD2 PHE A 564 51.773 25.320 58.259 1.00 25.83 C ATOM 230 CE1 PHE A 564 51.688 22.610 57.653 1.00 26.86 C ATOM 231 CE2 PHE A 564 51.208 24.894 57.059 1.00 26.23 C ATOM 232 CZ PHE A 564 51.166 23.538 56.757 1.00 26.71 C ATOM 233 N GLU A 565 53.160 25.289 63.596 1.00 25.20 N ATOM 234 CA GLU A 565 53.958 25.813 64.709 1.00 26.01 C ATOM 235 C GLU A 565 53.905 24.956 65.974 1.00 26.25 C ATOM 236 O GLU A 565 54.545 25.291 66.975 1.00 27.06 O ATOM 237 CB GLU A 565 53.493 27.229 65.065 1.00 26.43 C ATOM 238 CG GLU A 565 52.112 27.273 65.704 1.00 27.48 C ATOM 239 CD GLU A 565 51.007 27.612 64.719 1.00 29.85 C ATOM 240 OE1 GLU A 565 51.069 27.161 63.547 1.00 29.74 O ATOM 241 OE2 GLU A 565 50.062 28.322 65.128 1.00 29.41 O ATOM 242 N LEU A 566 53.157 23.856 65.932 1.00 25.62 N ATOM 243 CA LEU A 566 53.002 22.986 67.098 1.00 25.15 C ATOM 244 C LEU A 566 53.899 21.754 67.148 1.00 24.78 C ATOM 245 O LEU A 566 54.209 21.150 66.123 1.00 23.05 O ATOM 246 CB LEU A 566 51.543 22.518 67.214 1.00 25.60 C ATOM 247 CG LEU A 566 50.441 23.576 67.189 1.00 25.20 C ATOM 248 CD1 LEU A 566 49.075 22.889 67.203 1.00 25.03 C ATOM 249 CD2 LEU A 566 50.591 24.512 68.379 1.00 25.68 C ATOM 250 N SER A 567 54.298 21.382 68.363 1.00 24.50 N ATOM 251 CA SER A 567 55.127 20.198 68.577 1.00 24.64 C ATOM 252 C SER A 567 54.156 19.029 68.756 1.00 24.67 C ATOM 253 O SER A 567 52.952 19.238 68.906 1.00 22.93 O ATOM 254 CB SER A 567 55.948 20.348 69.857 1.00 24.05 C ATOM 255 OG SER A 567 55.087 20.298 70.982 1.00 22.98 O ATOM 256 N ASP A 568 54.668 17.805 68.747 1.00 25.45 N ATOM 257 CA ASP A 568 53.785 16.658 68.934 1.00 27.39 C ATOM 258 C ASP A 568 53.066 16.788 70.275 1.00 26.88 C ATOM 259 O ASP A 568 51.856 16.593 70.357 1.00 26.09 O ATOM 260 CB ASP A 568 54.570 15.343 68.888 1.00 28.31 C ATOM 261 CG ASP A 568 55.002 14.974 67.484 1.00 30.28 C ATOM 262 OD1 ASP A 568 54.551 15.634 66.525 1.00 31.20 O ATOM 263 OD2 ASP A 568 55.787 14.015 67.334 1.00 32.48 O ATOM 264 N LEU A 569 53.814 17.136 71.320 1.00 26.83 N ATOM 265 CA LEU A 569 53.221 17.292 72.645 1.00 27.03 C ATOM 266 C LEU A 569 52.036 18.245 72.612 1.00 26.05 C ATOM 267 O LEU A 569 50.993 17.966 73.198 1.00 25.43 O ATOM 268 CB LEU A 569 54.261 17.807 73.645 1.00 28.18 C ATOM 269 CG LEU A 569 53.750 18.206 75.037 1.00 29.46 C ATOM 270 CD1 LEU A 569 52.985 17.050 75.686 1.00 28.67 C ATOM 271 CD2 LEU A 569 54.944 18.608 75.907 1.00 30.57 C ATOM 272 N GLU A 570 52.196 19.365 71.915 1.00 25.43 N ATOM 273 CA GLU A 570 51.128 20.356 71.812 1.00 24.71 C ATOM 274 C GLU A 570 49.896 19.826 71.081 1.00 23.37 C ATOM 275 O GLU A 570 48.767 20.187 71.425 1.00 23.26 O ATOM 276 CB GLU A 570 51.636 21.628 71.115 1.00 25.60 C ATOM 277 CG GLU A 570 52.525 22.514 71.987 1.00 28.27 C ATOM 278 CD GLU A 570 53.057 23.728 71.244 1.00 28.00 C ATOM 279 OE1 GLU A 570 53.797 23.548 70.258 1.00 28.59 O ATOM 280 OE2 GLU A 570 52.734 24.864 71.642 1.00 30.03 O ATOM 281 N THR A 571 50.096 18.988 70.068 1.00 22.80 N ATOM 282 CA THR A 571 48.947 18.441 69.344 1.00 22.18 C ATOM 283 C THR A 571 48.197 17.480 70.261 1.00 22.78 C ATOM 284 O THR A 571 46.969 17.406 70.226 1.00 23.51 O ATOM 285 CB THR A 571 49.360 17.699 68.046 1.00 22.43 C ATOM 286 OG1 THR A 571 50.197 16.577 68.362 1.00 22.41 O ATOM 287 CG2 THR A 571 50.090 18.646 67.100 1.00 18.66 C ATOM 288 N ALA A 572 48.944 16.747 71.081 1.00 22.45 N ATOM 289 CA ALA A 572 48.350 15.811 72.035 1.00 23.41 C ATOM 290 C ALA A 572 47.502 16.580 73.055 1.00 23.81 C ATOM 291 O ALA A 572 46.420 16.138 73.434 1.00 24.50 O ATOM 292 CB ALA A 572 49.448 15.032 72.745 1.00 22.55 C ATOM 293 N LEU A 573 47.996 17.735 73.501 1.00 23.91 N ATOM 294 CA LEU A 573 47.242 18.551 74.453 1.00 23.75 C ATOM 295 C LEU A 573 45.977 19.077 73.792 1.00 22.73 C ATOM 296 O LEU A 573 44.933 19.164 74.430 1.00 23.52 O ATOM 297 CB LEU A 573 48.096 19.719 74.970 1.00 24.06 C ATOM 298 CG LEU A 573 49.316 19.301 75.805 1.00 25.24 C ATOM 299 CD1 LEU A 573 50.149 20.531 76.163 1.00 26.24 C ATOM 300 CD2 LEU A 573 48.850 18.587 77.074 1.00 25.62 C ATOM 301 N CYS A 574 46.069 19.428 72.510 1.00 22.65 N ATOM 302 CA CYS A 574 44.906 19.914 71.770 1.00 21.72 C ATOM 303 C CYS A 574 43.872 18.785 71.655 1.00 22.09 C ATOM 304 O CYS A 574 42.662 19.023 71.715 1.00 20.93 O ATOM 305 CB CYS A 574 45.302 20.353 70.356 1.00 22.58 C ATOM 306 SG CYS A 574 46.275 21.890 70.227 1.00 22.89 S ATOM 307 N THR A 575 44.352 17.564 71.462 1.00 21.45 N ATOM 308 CA THR A 575 43.451 16.413 71.333 1.00 22.28 C ATOM 309 C THR A 575 42.738 16.154 72.662 1.00 22.10 C ATOM 310 O THR A 575 41.563 15.809 72.681 1.00 24.22 O ATOM 311 CB THR A 575 44.225 15.153 70.884 1.00 21.93 C ATOM 312 OG1 THR A 575 44.863 15.422 69.629 1.00 20.68 O ATOM 313 CG2 THR A 575 43.265 13.951 70.709 1.00 21.15 C ATOM 314 N ILE A 576 43.443 16.322 73.775 1.00 23.78 N ATOM 315 CA ILE A 576 42.816 16.137 75.081 1.00 24.23 C ATOM 316 C ILE A 576 41.725 17.197 75.246 1.00 24.62 C ATOM 317 O ILE A 576 40.621 16.902 75.713 1.00 24.61 O ATOM 318 CB ILE A 576 43.837 16.283 76.238 1.00 24.90 C ATOM 319 CG1 ILE A 576 44.827 15.116 76.217 1.00 24.63 C ATOM 320 CG2 ILE A 576 43.110 16.307 77.580 1.00 24.33 C ATOM 321 CD1 ILE A 576 45.998 15.296 77.176 1.00 25.31 C ATOM 322 N ARG A 577 42.031 18.431 74.842 1.00 24.91 N ATOM 323 CA ARG A 577 41.068 19.521 74.959 1.00 24.32 C ATOM 324 C ARG A 577 39.808 19.243 74.156 1.00 24.61 C ATOM 325 O ARG A 577 38.702 19.601 74.575 1.00 24.50 O ATOM 326 CB ARG A 577 41.691 20.846 74.507 1.00 24.24 C ATOM 327 CG ARG A 577 40.707 22.007 74.481 1.00 25.24 C ATOM 328 CD ARG A 577 39.968 22.152 75.814 1.00 25.38 C ATOM 329 NE ARG A 577 40.892 22.368 76.923 1.00 26.44 N ATOM 330 CZ ARG A 577 40.524 22.480 78.196 1.00 26.97 C ATOM 331 NH1 ARG A 577 39.244 22.394 78.531 1.00 27.02 N ATOM 332 NH2 ARG A 577 41.440 22.686 79.134 1.00 27.69 N ATOM 333 N MET A 578 39.974 18.608 72.998 1.00 24.65 N ATOM 334 CA MET A 578 38.835 18.266 72.155 1.00 24.75 C ATOM 335 C MET A 578 37.923 17.305 72.920 1.00 24.47 C ATOM 336 O MET A 578 36.722 17.530 73.010 1.00 25.88 O ATOM 337 CB MET A 578 39.304 17.601 70.848 1.00 25.09 C ATOM 338 CG MET A 578 39.984 18.535 69.855 1.00 24.86 C ATOM 339 SD MET A 578 40.615 17.664 68.395 1.00 26.22 S ATOM 340 CE MET A 578 42.033 18.702 67.961 1.00 24.19 C ATOM 341 N PHE A 579 38.495 16.234 73.463 1.00 24.40 N ATOM 342 CA PHE A 579 37.713 15.253 74.222 1.00 25.06 C ATOM 343 C PHE A 579 37.057 15.896 75.437 1.00 25.72 C ATOM 344 O PHE A 579 35.903 15.613 75.770 1.00 26.35 O ATOM 345 CB PHE A 579 38.599 14.110 74.721 1.00 23.56 C ATOM 346 CG PHE A 579 38.871 13.051 73.693 1.00 24.34 C ATOM 347 CD1 PHE A 579 39.867 13.224 72.734 1.00 23.82 C ATOM 348 CD2 PHE A 579 38.128 11.875 73.686 1.00 23.12 C ATOM 349 CE1 PHE A 579 40.116 12.243 71.787 1.00 23.30 C ATOM 350 CE2 PHE A 579 38.369 10.889 72.743 1.00 22.91 C ATOM 351 CZ PHE A 579 39.365 11.071 71.793 1.00 23.86 C ATOM 352 N THR A 580 37.816 16.755 76.104 1.00 26.95 N ATOM 353 CA THR A 580 37.339 17.439 77.299 1.00 27.41 C ATOM 354 C THR A 580 36.173 18.376 77.029 1.00 28.05 C ATOM 355 O THR A 580 35.131 18.281 77.676 1.00 28.33 O ATOM 356 CB THR A 580 38.462 18.258 77.941 1.00 26.84 C ATOM 357 OG1 THR A 580 39.551 17.391 78.262 1.00 25.94 O ATOM 358 CG2 THR A 580 37.965 18.948 79.212 1.00 27.30 C ATOM 359 N ASP A 581 36.348 19.283 76.075 1.00 28.86 N ATOM 360 CA ASP A 581 35.303 20.249 75.768 1.00 29.73 C ATOM 361 C ASP A 581 34.059 19.656 75.129 1.00 30.39 C ATOM 362 O ASP A 581 32.982 20.253 75.205 1.00 30.81 O ATOM 363 CB ASP A 581 35.875 21.388 74.925 1.00 29.02 C ATOM 364 CG ASP A 581 36.610 22.419 75.775 1.00 30.46 C ATOM 365 OD1 ASP A 581 37.064 22.065 76.885 1.00 30.23 O ATOM 366 OD2 ASP A 581 36.740 23.579 75.336 1.00 30.07 O ATOM 367 N LEU A 582 34.193 18.489 74.503 1.00 30.71 N ATOM 368 CA LEU A 582 33.033 17.826 73.910 1.00 31.03 C ATOM 369 C LEU A 582 32.371 16.976 75.007 1.00 31.44 C ATOM 370 O LEU A 582 31.475 16.176 74.742 1.00 31.49 O ATOM 371 CB LEU A 582 33.451 16.942 72.726 1.00 30.86 C ATOM 372 CG LEU A 582 33.930 17.664 71.458 1.00 31.01 C ATOM 373 CD1 LEU A 582 34.478 16.654 70.461 1.00 29.77 C ATOM 374 CD2 LEU A 582 32.778 18.446 70.848 1.00 30.34 C ATOM 375 N ASN A 583 32.838 17.161 76.240 1.00 31.70 N ATOM 376 CA ASN A 583 32.312 16.458 77.408 1.00 32.47 C ATOM 377 C ASN A 583 32.455 14.938 77.385 1.00 32.09 C ATOM 378 O ASN A 583 31.783 14.238 78.146 1.00 32.03 O ATOM 379 CB ASN A 583 30.838 16.819 77.609 1.00 33.00 C ATOM 380 CG ASN A 583 30.625 18.307 77.749 1.00 34.05 C ATOM 381 OD1 ASN A 583 31.237 18.951 78.602 1.00 35.42 O ATOM 382 ND2 ASN A 583 29.758 18.865 76.913 1.00 33.48 N ATOM 383 N LEU A 584 33.337 14.432 76.531 1.00 31.46 N ATOM 384 CA LEU A 584 33.545 12.994 76.414 1.00 30.77 C ATOM 385 C LEU A 584 34.286 12.430 77.622 1.00 30.91 C ATOM 386 O LEU A 584 34.030 11.303 78.047 1.00 30.53 O ATOM 387 CB LEU A 584 34.306 12.686 75.122 1.00 30.25 C ATOM 388 CG LEU A 584 33.668 13.319 73.879 1.00 30.05 C ATOM 389 CD1 LEU A 584 34.515 13.021 72.639 1.00 28.59 C ATOM 390 CD2 LEU A 584 32.247 12.786 73.705 1.00 29.62 C ATOM 391 N VAL A 585 35.201 13.213 78.180 1.00 30.54 N ATOM 392 CA VAL A 585 35.948 12.766 79.346 1.00 31.44 C ATOM 393 C VAL A 585 35.009 12.619 80.543 1.00 32.33 C ATOM 394 O VAL A 585 35.147 11.686 81.328 1.00 31.86 O ATOM 395 CB VAL A 585 37.082 13.749 79.697 1.00 30.86 C ATOM 396 CG1 VAL A 585 37.716 13.371 81.033 1.00 31.14 C ATOM 397 CG2 VAL A 585 38.134 13.727 78.595 1.00 30.70 C ATOM 398 N GLN A 586 34.050 13.534 80.667 1.00 33.63 N ATOM 399 CA GLN A 586 33.081 13.496 81.764 1.00 35.21 C ATOM 400 C GLN A 586 31.989 12.457 81.530 1.00 34.88 C ATOM 401 O GLN A 586 31.774 11.577 82.359 1.00 35.41 O ATOM 402 CB GLN A 586 32.407 14.864 81.949 1.00 37.53 C ATOM 403 CG GLN A 586 31.369 14.893 83.083 1.00 41.13 C ATOM 404 CD GLN A 586 30.398 16.065 82.978 1.00 43.71 C ATOM 405 OE1 GLN A 586 29.729 16.237 81.954 1.00 45.81 O ATOM 406 NE2 GLN A 586 30.308 16.872 84.041 1.00 44.18 N ATOM 407 N ASN A 587 31.300 12.570 80.398 1.00 34.50 N ATOM 408 CA ASN A 587 30.204 11.669 80.075 1.00 34.82 C ATOM 409 C ASN A 587 30.551 10.189 79.998 1.00 34.80 C ATOM 410 O ASN A 587 29.670 9.345 80.135 1.00 34.47 O ATOM 411 CB ASN A 587 29.527 12.094 78.768 1.00 35.34 C ATOM 412 CG ASN A 587 28.727 13.379 78.917 1.00 36.34 C ATOM 413 OD1 ASN A 587 28.496 13.855 80.028 1.00 36.84 O ATOM 414 ND2 ASN A 587 28.292 13.937 77.798 1.00 35.75 N ATOM 415 N PHE A 588 31.822 9.866 79.789 1.00 33.94 N ATOM 416 CA PHE A 588 32.217 8.468 79.692 1.00 33.99 C ATOM 417 C PHE A 588 33.326 8.100 80.656 1.00 33.96 C ATOM 418 O PHE A 588 34.027 7.104 80.479 1.00 33.81 O ATOM 419 CB PHE A 588 32.589 8.157 78.244 1.00 32.61 C ATOM 420 CG PHE A 588 31.454 8.387 77.292 1.00 32.74 C ATOM 421 CD1 PHE A 588 30.308 7.596 77.363 1.00 31.91 C ATOM 422 CD2 PHE A 588 31.490 9.432 76.376 1.00 31.91 C ATOM 423 CE1 PHE A 588 29.216 7.844 76.539 1.00 31.46 C ATOM 424 CE2 PHE A 588 30.397 9.689 75.545 1.00 32.32 C ATOM 425 CZ PHE A 588 29.259 8.893 75.630 1.00 32.13 C ATOM 426 N GLN A 589 33.459 8.927 81.686 1.00 35.48 N ATOM 427 CA GLN A 589 34.435 8.742 82.754 1.00 36.24 C ATOM 428 C GLN A 589 35.788 8.208 82.305 1.00 35.50 C ATOM 429 O GLN A 589 36.230 7.153 82.766 1.00 35.43 O ATOM 430 CB GLN A 589 33.848 7.807 83.814 1.00 38.75 C ATOM 431 CG GLN A 589 32.329 7.870 83.898 1.00 42.30 C ATOM 432 CD GLN A 589 31.752 6.903 84.911 1.00 45.65 C ATOM 433 OE1 GLN A 589 30.561 6.583 84.868 1.00 47.58 O ATOM 434 NE2 GLN A 589 32.589 6.437 85.840 1.00 46.23 N ATOM 435 N MET A 590 36.461 8.934 81.421 1.00 34.15 N ATOM 436 CA MET A 590 37.769 8.491 80.958 1.00 33.36 C ATOM 437 C MET A 590 38.833 8.905 81.966 1.00 33.32 C ATOM 438 O MET A 590 38.819 10.032 82.465 1.00 33.23 O ATOM 439 CB MET A 590 38.109 9.116 79.601 1.00 33.37 C ATOM 440 CG MET A 590 37.144 8.793 78.476 1.00 33.12 C ATOM 441 SD MET A 590 37.616 9.632 76.950 1.00 31.44 S ATOM 442 CE MET A 590 36.284 9.164 75.875 1.00 31.30 C ATOM 443 N LYS A 591 39.753 8.001 82.274 1.00 32.69 N ATOM 444 CA LYS A 591 40.823 8.342 83.196 1.00 33.22 C ATOM 445 C LYS A 591 41.882 9.104 82.403 1.00 32.29 C ATOM 446 O LYS A 591 42.205 8.736 81.267 1.00 31.27 O ATOM 447 CB LYS A 591 41.417 7.084 83.827 1.00 34.29 C ATOM 448 CG LYS A 591 40.457 6.387 84.784 1.00 36.87 C ATOM 449 CD LYS A 591 41.173 5.355 85.641 1.00 38.65 C ATOM 450 CE LYS A 591 40.225 4.741 86.660 1.00 39.80 C ATOM 451 NZ LYS A 591 40.936 3.829 87.597 1.00 40.74 N ATOM 452 N HIS A 592 42.419 10.161 83.002 1.00 31.35 N ATOM 453 CA HIS A 592 43.409 10.995 82.331 1.00 30.69 C ATOM 454 C HIS A 592 44.564 10.231 81.713 1.00 30.36 C ATOM 455 O HIS A 592 44.873 10.419 80.536 1.00 30.09 O ATOM 456 CB HIS A 592 43.974 12.040 83.293 1.00 31.16 C ATOM 457 CG HIS A 592 44.789 13.100 82.618 1.00 31.18 C ATOM 458 ND1 HIS A 592 44.241 14.009 81.738 1.00 31.30 N ATOM 459 CD2 HIS A 592 46.108 13.399 82.695 1.00 31.66 C ATOM 460 CE1 HIS A 592 45.186 14.825 81.304 1.00 32.27 C ATOM 461 NE2 HIS A 592 46.329 14.477 81.869 1.00 32.22 N ATOM 462 N GLU A 593 45.205 9.374 82.502 1.00 30.02 N ATOM 463 CA GLU A 593 46.349 8.617 82.013 1.00 29.94 C ATOM 464 C GLU A 593 45.999 7.690 80.855 1.00 28.34 C ATOM 465 O GLU A 593 46.826 7.458 79.981 1.00 27.62 O ATOM 466 CB GLU A 593 46.990 7.806 83.148 1.00 31.81 C ATOM 467 CG GLU A 593 46.037 6.850 83.839 1.00 35.34 C ATOM 468 CD GLU A 593 45.399 7.452 85.080 1.00 37.30 C ATOM 469 OE1 GLU A 593 45.005 8.641 85.051 1.00 37.55 O ATOM 470 OE2 GLU A 593 45.284 6.723 86.087 1.00 39.65 O ATOM 471 N VAL A 594 44.779 7.156 80.857 1.00 27.30 N ATOM 472 CA VAL A 594 44.329 6.256 79.793 1.00 25.58 C ATOM 473 C VAL A 594 44.148 7.025 78.480 1.00 25.20 C ATOM 474 O VAL A 594 44.601 6.588 77.418 1.00 24.16 O ATOM 475 CB VAL A 594 42.990 5.576 80.169 1.00 26.28 C ATOM 476 CG1 VAL A 594 42.499 4.703 79.016 1.00 25.68 C ATOM 477 CG2 VAL A 594 43.174 4.733 81.440 1.00 25.44 C ATOM 478 N LEU A 595 43.478 8.168 78.558 1.00 24.80 N ATOM 479 CA LEU A 595 43.259 9.003 77.385 1.00 25.06 C ATOM 480 C LEU A 595 44.602 9.449 76.802 1.00 25.24 C ATOM 481 O LEU A 595 44.793 9.448 75.584 1.00 25.32 O ATOM 482 CB LEU A 595 42.413 10.225 77.759 1.00 24.44 C ATOM 483 CG LEU A 595 42.164 11.235 76.633 1.00 24.77 C ATOM 484 CD1 LEU A 595 41.654 10.515 75.395 1.00 24.53 C ATOM 485 CD2 LEU A 595 41.162 12.279 77.093 1.00 23.71 C ATOM 486 N CYS A 596 45.532 9.822 77.680 1.00 25.65 N ATOM 487 CA CYS A 596 46.866 10.258 77.264 1.00 26.25 C ATOM 488 C CYS A 596 47.615 9.144 76.543 1.00 27.05 C ATOM 489 O CYS A 596 48.216 9.358 75.481 1.00 26.85 O ATOM 490 CB CYS A 596 47.696 10.698 78.478 1.00 25.96 C ATOM 491 SG CYS A 596 47.267 12.320 79.167 1.00 26.65 S ATOM 492 N ARG A 597 47.597 7.959 77.144 1.00 26.45 N ATOM 493 CA ARG A 597 48.269 6.804 76.572 1.00 26.81 C ATOM 494 C ARG A 597 47.652 6.487 75.207 1.00 25.59 C ATOM 495 O ARG A 597 48.368 6.245 74.242 1.00 25.07 O ATOM 496 CB ARG A 597 48.128 5.607 77.520 1.00 28.07 C ATOM 497 CG ARG A 597 49.129 4.498 77.292 1.00 33.12 C ATOM 498 CD ARG A 597 49.106 3.508 78.452 1.00 36.48 C ATOM 499 NE ARG A 597 49.348 4.178 79.727 1.00 40.06 N ATOM 500 CZ ARG A 597 49.240 3.594 80.917 1.00 41.68 C ATOM 501 NH1 ARG A 597 48.893 2.319 81.002 1.00 43.80 N ATOM 502 NH2 ARG A 597 49.475 4.284 82.025 1.00 42.94 N ATOM 503 N TRP A 598 46.323 6.508 75.134 1.00 24.05 N ATOM 504 CA TRP A 598 45.611 6.229 73.891 1.00 23.45 C ATOM 505 C TRP A 598 46.018 7.206 72.784 1.00 23.31 C ATOM 506 O TRP A 598 46.324 6.800 71.665 1.00 23.07 O ATOM 507 CB TRP A 598 44.097 6.327 74.109 1.00 23.98 C ATOM 508 CG TRP A 598 43.306 6.013 72.866 1.00 24.18 C ATOM 509 CD1 TRP A 598 43.022 4.776 72.362 1.00 24.39 C ATOM 510 CD2 TRP A 598 42.763 6.957 71.937 1.00 24.15 C ATOM 511 NE1 TRP A 598 42.339 4.892 71.175 1.00 24.43 N ATOM 512 CE2 TRP A 598 42.170 6.222 70.891 1.00 24.35 C ATOM 513 CE3 TRP A 598 42.726 8.358 71.888 1.00 23.61 C ATOM 514 CZ2 TRP A 598 41.547 6.838 69.802 1.00 24.16 C ATOM 515 CZ3 TRP A 598 42.106 8.968 70.806 1.00 24.29 C ATOM 516 CH2 TRP A 598 41.525 8.206 69.779 1.00 24.39 C ATOM 517 N ILE A 599 46.013 8.496 73.103 1.00 23.06 N ATOM 518 CA ILE A 599 46.386 9.515 72.135 1.00 22.60 C ATOM 519 C ILE A 599 47.799 9.254 71.630 1.00 22.84 C ATOM 520 O ILE A 599 48.060 9.337 70.429 1.00 21.66 O ATOM 521 CB ILE A 599 46.311 10.926 72.763 1.00 22.65 C ATOM 522 CG1 ILE A 599 44.849 11.266 73.063 1.00 22.83 C ATOM 523 CG2 ILE A 599 46.948 11.960 71.826 1.00 23.20 C ATOM 524 CD1 ILE A 599 44.645 12.548 73.848 1.00 21.99 C ATOM 525 N LEU A 600 48.707 8.919 72.545 1.00 21.91 N ATOM 526 CA LEU A 600 50.085 8.653 72.152 1.00 21.71 C ATOM 527 C LEU A 600 50.233 7.379 71.319 1.00 21.76 C ATOM 528 O LEU A 600 51.101 7.315 70.448 1.00 20.96 O ATOM 529 CB LEU A 600 50.994 8.598 73.385 1.00 23.21 C ATOM 530 CG LEU A 600 51.138 9.936 74.130 1.00 23.86 C ATOM 531 CD1 LEU A 600 52.034 9.746 75.343 1.00 25.69 C ATOM 532 CD2 LEU A 600 51.709 11.012 73.207 1.00 23.50 C ATOM 533 N SER A 601 49.406 6.365 71.584 1.00 20.67 N ATOM 534 CA SER A 601 49.469 5.134 70.789 1.00 21.19 C ATOM 535 C SER A 601 48.963 5.466 69.383 1.00 20.71 C ATOM 536 O SER A 601 49.533 5.030 68.384 1.00 21.98 O ATOM 537 CB SER A 601 48.591 4.024 71.398 1.00 20.80 C ATOM 538 OG SER A 601 49.078 3.606 72.659 1.00 21.54 O ATOM 539 N VAL A 602 47.889 6.245 69.306 1.00 20.80 N ATOM 540 CA VAL A 602 47.344 6.633 68.009 1.00 20.64 C ATOM 541 C VAL A 602 48.426 7.366 67.224 1.00 20.78 C ATOM 542 O VAL A 602 48.758 6.983 66.103 1.00 20.16 O ATOM 543 CB VAL A 602 46.107 7.548 68.165 1.00 20.34 C ATOM 544 CG1 VAL A 602 45.777 8.210 66.829 1.00 19.39 C ATOM 545 CG2 VAL A 602 44.908 6.723 68.655 1.00 19.68 C ATOM 546 N LYS A 603 48.991 8.412 67.819 1.00 21.10 N ATOM 547 CA LYS A 603 50.037 9.178 67.148 1.00 22.43 C ATOM 548 C LYS A 603 51.185 8.275 66.690 1.00 22.67 C ATOM 549 O LYS A 603 51.657 8.366 65.560 1.00 22.58 O ATOM 550 CB LYS A 603 50.587 10.266 68.082 1.00 23.15 C ATOM 551 CG LYS A 603 51.634 11.158 67.415 1.00 24.97 C ATOM 552 CD LYS A 603 52.306 12.107 68.392 1.00 27.74 C ATOM 553 CE LYS A 603 53.136 11.321 69.391 1.00 28.43 C ATOM 554 NZ LYS A 603 54.282 12.075 69.918 1.00 30.45 N ATOM 555 N LYS A 604 51.633 7.404 67.582 1.00 24.48 N ATOM 556 CA LYS A 604 52.731 6.492 67.278 1.00 26.81 C ATOM 557 C LYS A 604 52.446 5.564 66.092 1.00 26.73 C ATOM 558 O LYS A 604 53.370 5.100 65.420 1.00 26.36 O ATOM 559 CB LYS A 604 53.048 5.652 68.520 1.00 28.66 C ATOM 560 CG LYS A 604 54.159 4.644 68.337 1.00 32.77 C ATOM 561 CD LYS A 604 54.322 3.789 69.587 1.00 34.54 C ATOM 562 CE LYS A 604 55.359 2.691 69.371 1.00 37.21 C ATOM 563 NZ LYS A 604 55.588 1.902 70.618 1.00 38.86 N ATOM 564 N ASN A 605 51.172 5.303 65.818 1.00 26.83 N ATOM 565 CA ASN A 605 50.841 4.401 64.727 1.00 27.59 C ATOM 566 C ASN A 605 50.622 5.007 63.342 1.00 27.35 C ATOM 567 O ASN A 605 50.079 4.360 62.440 1.00 27.36 O ATOM 568 CB ASN A 605 49.681 3.496 65.145 1.00 28.86 C ATOM 569 CG ASN A 605 50.163 2.321 65.987 1.00 31.06 C ATOM 570 OD1 ASN A 605 50.746 1.369 65.462 1.00 31.33 O ATOM 571 ND2 ASN A 605 49.960 2.403 67.301 1.00 32.35 N ATOM 572 N TYR A 606 51.048 6.255 63.183 1.00 25.91 N ATOM 573 CA TYR A 606 51.013 6.910 61.885 1.00 25.27 C ATOM 574 C TYR A 606 52.502 6.944 61.535 1.00 26.48 C ATOM 575 O TYR A 606 53.342 6.924 62.437 1.00 25.39 O ATOM 576 CB TYR A 606 50.448 8.332 61.974 1.00 22.38 C ATOM 577 CG TYR A 606 48.933 8.374 61.938 1.00 21.11 C ATOM 578 CD1 TYR A 606 48.183 8.394 63.113 1.00 19.86 C ATOM 579 CD2 TYR A 606 48.249 8.340 60.721 1.00 20.07 C ATOM 580 CE1 TYR A 606 46.785 8.375 63.077 1.00 19.67 C ATOM 581 CE2 TYR A 606 46.861 8.318 60.672 1.00 18.50 C ATOM 582 CZ TYR A 606 46.132 8.335 61.849 1.00 20.00 C ATOM 583 OH TYR A 606 44.750 8.319 61.789 1.00 19.86 O ATOM 584 N ARG A 607 52.836 6.969 60.249 1.00 27.83 N ATOM 585 CA ARG A 607 54.238 6.991 59.839 1.00 30.73 C ATOM 586 C ARG A 607 54.640 8.401 59.414 1.00 31.88 C ATOM 587 O ARG A 607 54.146 8.931 58.417 1.00 32.06 O ATOM 588 CB ARG A 607 54.458 5.995 58.703 1.00 31.85 C ATOM 589 CG ARG A 607 54.226 4.551 59.130 1.00 33.74 C ATOM 590 CD ARG A 607 53.840 3.673 57.957 1.00 35.36 C ATOM 591 NE ARG A 607 53.511 2.317 58.386 1.00 36.57 N ATOM 592 CZ ARG A 607 54.409 1.413 58.760 1.00 37.51 C ATOM 593 NH1 ARG A 607 55.702 1.712 58.751 1.00 38.76 N ATOM 594 NH2 ARG A 607 54.013 0.212 59.158 1.00 37.98 N ATOM 595 N LYS A 608 55.542 9.003 60.176 1.00 33.30 N ATOM 596 CA LYS A 608 55.976 10.364 59.892 1.00 35.64 C ATOM 597 C LYS A 608 56.750 10.502 58.586 1.00 35.56 C ATOM 598 O LYS A 608 56.875 11.605 58.052 1.00 36.10 O ATOM 599 CB LYS A 608 56.801 10.912 61.064 1.00 37.50 C ATOM 600 CG LYS A 608 57.010 12.427 61.007 1.00 40.44 C ATOM 601 CD LYS A 608 57.539 12.989 62.329 1.00 41.67 C ATOM 602 CE LYS A 608 57.666 14.512 62.256 1.00 43.01 C ATOM 603 NZ LYS A 608 58.129 15.120 63.542 1.00 44.18 N ATOM 604 N ASN A 609 57.262 9.391 58.064 1.00 35.19 N ATOM 605 CA ASN A 609 58.003 9.431 56.809 1.00 34.61 C ATOM 606 C ASN A 609 57.044 9.651 55.635 1.00 33.47 C ATOM 607 O ASN A 609 57.474 10.023 54.548 1.00 33.88 O ATOM 608 CB ASN A 609 58.792 8.129 56.599 1.00 36.07 C ATOM 609 CG ASN A 609 57.889 6.934 56.353 1.00 37.51 C ATOM 610 OD1 ASN A 609 56.994 6.655 57.142 1.00 38.35 O ATOM 611 ND2 ASN A 609 58.122 6.225 55.252 1.00 38.24 N ATOM 612 N VAL A 610 55.750 9.418 55.851 1.00 31.24 N ATOM 613 CA VAL A 610 54.755 9.619 54.795 1.00 29.70 C ATOM 614 C VAL A 610 54.379 11.105 54.765 1.00 29.34 C ATOM 615 O VAL A 610 53.872 11.648 55.751 1.00 28.89 O ATOM 616 CB VAL A 610 53.499 8.749 55.043 1.00 29.17 C ATOM 617 CG1 VAL A 610 52.449 9.031 53.991 1.00 27.99 C ATOM 618 CG2 VAL A 610 53.886 7.276 55.021 1.00 28.13 C ATOM 619 N ALA A 611 54.633 11.750 53.626 1.00 28.19 N ATOM 620 CA ALA A 611 54.396 13.186 53.448 1.00 26.86 C ATOM 621 C ALA A 611 53.063 13.772 53.900 1.00 25.97 C ATOM 622 O ALA A 611 53.042 14.786 54.605 1.00 26.34 O ATOM 623 CB ALA A 611 54.654 13.573 51.995 1.00 26.87 C ATOM 624 N TYR A 612 51.951 13.171 53.492 1.00 24.90 N ATOM 625 CA TYR A 612 50.654 13.704 53.887 1.00 24.31 C ATOM 626 C TYR A 612 49.854 12.833 54.845 1.00 23.26 C ATOM 627 O TYR A 612 49.371 13.318 55.863 1.00 22.10 O ATOM 628 CB TYR A 612 49.782 14.010 52.666 1.00 24.87 C ATOM 629 CG TYR A 612 48.521 14.742 53.060 1.00 26.36 C ATOM 630 CD1 TYR A 612 48.593 15.991 53.675 1.00 26.89 C ATOM 631 CD2 TYR A 612 47.265 14.159 52.898 1.00 27.39 C ATOM 632 CE1 TYR A 612 47.450 16.640 54.130 1.00 27.55 C ATOM 633 CE2 TYR A 612 46.111 14.802 53.351 1.00 28.71 C ATOM 634 CZ TYR A 612 46.217 16.042 53.970 1.00 28.91 C ATOM 635 OH TYR A 612 45.093 16.678 54.455 1.00 31.34 O ATOM 636 N HIS A 613 49.691 11.555 54.518 1.00 23.09 N ATOM 637 CA HIS A 613 48.933 10.667 55.395 1.00 22.38 C ATOM 638 C HIS A 613 49.753 10.310 56.631 1.00 22.61 C ATOM 639 O HIS A 613 50.366 9.249 56.708 1.00 22.98 O ATOM 640 CB HIS A 613 48.511 9.404 54.641 1.00 21.97 C ATOM 641 CG HIS A 613 47.416 9.640 53.646 1.00 22.49 C ATOM 642 ND1 HIS A 613 47.626 9.621 52.284 1.00 21.52 N ATOM 643 CD2 HIS A 613 46.108 9.948 53.819 1.00 22.36 C ATOM 644 CE1 HIS A 613 46.498 9.910 51.661 1.00 22.32 C ATOM 645 NE2 HIS A 613 45.560 10.112 52.569 1.00 23.90 N ATOM 646 N ASN A 614 49.753 11.218 57.601 1.00 21.77 N ATOM 647 CA ASN A 614 50.501 11.030 58.839 1.00 21.48 C ATOM 648 C ASN A 614 49.682 11.561 60.015 1.00 20.79 C ATOM 649 O ASN A 614 48.537 11.981 59.840 1.00 21.10 O ATOM 650 CB ASN A 614 51.846 11.764 58.743 1.00 21.40 C ATOM 651 CG ASN A 614 51.696 13.202 58.276 1.00 22.32 C ATOM 652 OD1 ASN A 614 50.932 13.971 58.850 1.00 23.24 O ATOM 653 ND2 ASN A 614 52.431 13.571 57.228 1.00 22.85 N ATOM 654 N TRP A 615 50.269 11.551 61.204 1.00 19.62 N ATOM 655 CA TRP A 615 49.573 12.021 62.393 1.00 19.39 C ATOM 656 C TRP A 615 48.991 13.432 62.268 1.00 19.87 C ATOM 657 O TRP A 615 47.849 13.676 62.676 1.00 19.30 O ATOM 658 CB TRP A 615 50.510 11.961 63.602 1.00 20.53 C ATOM 659 CG TRP A 615 50.076 12.824 64.757 1.00 21.49 C ATOM 660 CD1 TRP A 615 50.721 13.932 65.240 1.00 21.63 C ATOM 661 CD2 TRP A 615 48.897 12.667 65.556 1.00 21.69 C ATOM 662 NE1 TRP A 615 50.016 14.470 66.289 1.00 21.68 N ATOM 663 CE2 TRP A 615 48.893 13.714 66.504 1.00 22.41 C ATOM 664 CE3 TRP A 615 47.844 11.746 65.564 1.00 22.46 C ATOM 665 CZ2 TRP A 615 47.873 13.862 67.452 1.00 21.99 C ATOM 666 CZ3 TRP A 615 46.830 11.896 66.507 1.00 22.69 C ATOM 667 CH2 TRP A 615 46.854 12.946 67.436 1.00 21.31 C ATOM 668 N ARG A 616 49.765 14.360 61.712 1.00 19.16 N ATOM 669 CA ARG A 616 49.279 15.732 61.583 1.00 19.38 C ATOM 670 C ARG A 616 48.003 15.815 60.750 1.00 18.88 C ATOM 671 O ARG A 616 47.126 16.626 61.035 1.00 20.14 O ATOM 672 CB ARG A 616 50.372 16.636 61.006 1.00 19.58 C ATOM 673 CG ARG A 616 51.601 16.777 61.939 1.00 19.33 C ATOM 674 CD ARG A 616 51.205 17.143 63.382 1.00 19.19 C ATOM 675 NE ARG A 616 52.370 17.272 64.264 1.00 20.83 N ATOM 676 CZ ARG A 616 52.864 18.429 64.708 1.00 21.16 C ATOM 677 NH1 ARG A 616 52.295 19.579 64.363 1.00 20.72 N ATOM 678 NH2 ARG A 616 53.943 18.437 65.481 1.00 20.65 N ATOM 679 N HIS A 617 47.891 14.978 59.726 1.00 19.11 N ATOM 680 CA HIS A 617 46.680 14.973 58.914 1.00 18.93 C ATOM 681 C HIS A 617 45.513 14.494 59.778 1.00 18.83 C ATOM 682 O HIS A 617 44.438 15.093 59.775 1.00 19.75 O ATOM 683 CB HIS A 617 46.836 14.050 57.699 1.00 18.84 C ATOM 684 CG HIS A 617 45.531 13.665 57.077 1.00 19.13 C ATOM 685 ND1 HIS A 617 44.626 14.595 56.614 1.00 18.00 N ATOM 686 CD2 HIS A 617 44.960 12.453 56.891 1.00 19.38 C ATOM 687 CE1 HIS A 617 43.549 13.971 56.172 1.00 19.86 C ATOM 688 NE2 HIS A 617 43.727 12.671 56.330 1.00 19.30 N ATOM 689 N ALA A 618 45.732 13.412 60.520 1.00 19.43 N ATOM 690 CA ALA A 618 44.693 12.849 61.378 1.00 19.60 C ATOM 691 C ALA A 618 44.286 13.857 62.448 1.00 19.42 C ATOM 692 O ALA A 618 43.102 14.058 62.716 1.00 18.64 O ATOM 693 CB ALA A 618 45.192 11.566 62.027 1.00 20.51 C ATOM 694 N PHE A 619 45.282 14.482 63.063 1.00 19.50 N ATOM 695 CA PHE A 619 45.045 15.487 64.088 1.00 20.02 C ATOM 696 C PHE A 619 44.228 16.632 63.489 1.00 20.28 C ATOM 697 O PHE A 619 43.252 17.081 64.076 1.00 21.04 O ATOM 698 CB PHE A 619 46.385 16.016 64.620 1.00 20.99 C ATOM 699 CG PHE A 619 46.247 17.208 65.522 1.00 20.46 C ATOM 700 CD1 PHE A 619 45.570 17.105 66.735 1.00 20.41 C ATOM 701 CD2 PHE A 619 46.781 18.442 65.152 1.00 20.31 C ATOM 702 CE1 PHE A 619 45.423 18.215 67.573 1.00 20.49 C ATOM 703 CE2 PHE A 619 46.640 19.560 65.980 1.00 20.02 C ATOM 704 CZ PHE A 619 45.960 19.447 67.193 1.00 19.64 C ATOM 705 N ASN A 620 44.627 17.099 62.311 1.00 20.76 N ATOM 706 CA ASN A 620 43.901 18.172 61.644 1.00 21.16 C ATOM 707 C ASN A 620 42.464 17.770 61.301 1.00 21.75 C ATOM 708 O ASN A 620 41.541 18.580 61.407 1.00 21.18 O ATOM 709 CB ASN A 620 44.652 18.605 60.382 1.00 21.66 C ATOM 710 CG ASN A 620 45.707 19.659 60.678 1.00 22.26 C ATOM 711 OD1 ASN A 620 45.397 20.698 61.256 1.00 23.57 O ATOM 712 ND2 ASN A 620 46.947 19.396 60.296 1.00 22.73 N ATOM 713 N THR A 621 42.273 16.517 60.898 1.00 21.37 N ATOM 714 CA THR A 621 40.932 16.031 60.562 1.00 21.07 C ATOM 715 C THR A 621 40.039 16.095 61.799 1.00 20.78 C ATOM 716 O THR A 621 38.871 16.485 61.717 1.00 21.07 O ATOM 717 CB THR A 621 40.978 14.573 60.043 1.00 21.12 C ATOM 718 OG1 THR A 621 41.777 14.522 58.857 1.00 21.30 O ATOM 719 CG2 THR A 621 39.564 14.063 59.705 1.00 20.92 C ATOM 720 N ALA A 622 40.596 15.719 62.945 1.00 20.37 N ATOM 721 CA ALA A 622 39.858 15.740 64.204 1.00 20.73 C ATOM 722 C ALA A 622 39.576 17.180 64.637 1.00 21.19 C ATOM 723 O ALA A 622 38.494 17.495 65.122 1.00 20.84 O ATOM 724 CB ALA A 622 40.651 15.015 65.281 1.00 19.81 C ATOM 725 N GLN A 623 40.556 18.057 64.461 1.00 21.37 N ATOM 726 CA GLN A 623 40.366 19.454 64.829 1.00 21.92 C ATOM 727 C GLN A 623 39.200 20.020 64.017 1.00 21.79 C ATOM 728 O GLN A 623 38.322 20.694 64.563 1.00 21.48 O ATOM 729 CB GLN A 623 41.655 20.244 64.570 1.00 22.41 C ATOM 730 CG GLN A 623 41.589 21.723 64.946 1.00 23.04 C ATOM 731 CD GLN A 623 40.895 22.547 63.888 1.00 23.30 C ATOM 732 OE1 GLN A 623 41.096 22.326 62.698 1.00 23.16 O ATOM 733 NE2 GLN A 623 40.088 23.510 64.312 1.00 21.67 N ATOM 734 N CYS A 624 39.172 19.721 62.722 1.00 21.83 N ATOM 735 CA CYS A 624 38.091 20.207 61.877 1.00 23.04 C ATOM 736 C CYS A 624 36.755 19.633 62.376 1.00 23.65 C ATOM 737 O CYS A 624 35.738 20.316 62.363 1.00 23.51 O ATOM 738 CB CYS A 624 38.333 19.813 60.419 1.00 22.98 C ATOM 739 SG CYS A 624 37.111 20.472 59.244 1.00 23.32 S ATOM 740 N MET A 625 36.760 18.381 62.827 1.00 24.06 N ATOM 741 CA MET A 625 35.529 17.772 63.335 1.00 24.37 C ATOM 742 C MET A 625 35.079 18.569 64.559 1.00 24.89 C ATOM 743 O MET A 625 33.901 18.894 64.713 1.00 24.29 O ATOM 744 CB MET A 625 35.775 16.307 63.728 1.00 24.65 C ATOM 745 CG MET A 625 34.537 15.552 64.236 1.00 23.88 C ATOM 746 SD MET A 625 33.241 15.329 62.988 1.00 24.24 S ATOM 747 CE MET A 625 34.043 14.138 61.870 1.00 23.38 C ATOM 748 N PHE A 626 36.035 18.882 65.426 1.00 25.12 N ATOM 749 CA PHE A 626 35.755 19.642 66.635 1.00 25.63 C ATOM 750 C PHE A 626 35.176 21.006 66.269 1.00 25.87 C ATOM 751 O PHE A 626 34.162 21.429 66.829 1.00 25.20 O ATOM 752 CB PHE A 626 37.038 19.817 67.449 1.00 25.83 C ATOM 753 CG PHE A 626 36.848 20.587 68.723 1.00 27.11 C ATOM 754 CD1 PHE A 626 36.312 19.971 69.851 1.00 26.38 C ATOM 755 CD2 PHE A 626 37.199 21.936 68.793 1.00 26.52 C ATOM 756 CE1 PHE A 626 36.125 20.688 71.037 1.00 27.69 C ATOM 757 CE2 PHE A 626 37.017 22.664 69.975 1.00 28.07 C ATOM 758 CZ PHE A 626 36.478 22.037 71.099 1.00 27.37 C ATOM 759 N ALA A 627 35.819 21.683 65.319 1.00 25.94 N ATOM 760 CA ALA A 627 35.365 22.997 64.870 1.00 26.64 C ATOM 761 C ALA A 627 33.977 22.916 64.242 1.00 27.82 C ATOM 762 O ALA A 627 33.135 23.788 64.462 1.00 27.51 O ATOM 763 CB ALA A 627 36.360 23.587 63.869 1.00 27.41 C ATOM 764 N ALA A 628 33.729 21.870 63.461 1.00 28.11 N ATOM 765 CA ALA A 628 32.427 21.726 62.827 1.00 28.84 C ATOM 766 C ALA A 628 31.352 21.491 63.893 1.00 30.25 C ATOM 767 O ALA A 628 30.215 21.954 63.752 1.00 30.53 O ATOM 768 CB ALA A 628 32.454 20.583 61.822 1.00 29.11 C ATOM 769 N LEU A 629 31.720 20.790 64.965 1.00 31.21 N ATOM 770 CA LEU A 629 30.797 20.514 66.065 1.00 31.99 C ATOM 771 C LEU A 629 30.530 21.770 66.892 1.00 32.80 C ATOM 772 O LEU A 629 29.388 22.052 67.251 1.00 32.42 O ATOM 773 CB LEU A 629 31.361 19.429 66.988 1.00 31.44 C ATOM 774 CG LEU A 629 31.436 17.993 66.462 1.00 32.76 C ATOM 775 CD1 LEU A 629 32.178 17.119 67.464 1.00 31.09 C ATOM 776 CD2 LEU A 629 30.031 17.462 66.224 1.00 32.65 C ATOM 777 N LYS A 630 31.592 22.505 67.210 1.00 32.93 N ATOM 778 CA LYS A 630 31.476 23.732 67.994 1.00 34.28 C ATOM 779 C LYS A 630 31.076 24.911 67.112 1.00 34.39 C ATOM 780 O LYS A 630 29.894 25.213 66.964 1.00 34.34 O ATOM 781 CB LYS A 630 32.802 24.051 68.690 1.00 34.70 C ATOM 782 CG LYS A 630 33.190 23.064 69.767 1.00 35.41 C ATOM 783 CD LYS A 630 32.153 23.027 70.875 1.00 36.36 C ATOM 784 CE LYS A 630 32.603 22.114 71.996 1.00 37.50 C ATOM 785 NZ LYS A 630 31.620 22.046 73.113 1.00 39.26 N ATOM 786 N ALA A 631 32.071 25.563 66.520 1.00 34.00 N ATOM 787 CA ALA A 631 31.828 26.708 65.655 1.00 34.98 C ATOM 788 C ALA A 631 30.745 26.411 64.615 1.00 35.56 C ATOM 789 O ALA A 631 29.884 27.250 64.350 1.00 35.81 O ATOM 790 CB ALA A 631 33.127 27.124 64.967 1.00 34.27 C ATOM 791 N GLY A 632 30.784 25.216 64.034 1.00 35.65 N ATOM 792 CA GLY A 632 29.795 24.848 63.036 1.00 36.45 C ATOM 793 C GLY A 632 28.444 24.454 63.614 1.00 37.05 C ATOM 794 O GLY A 632 27.514 24.153 62.870 1.00 36.87 O ATOM 795 N LYS A 633 28.343 24.443 64.939 1.00 38.34 N ATOM 796 CA LYS A 633 27.104 24.101 65.640 1.00 40.04 C ATOM 797 C LYS A 633 26.452 22.772 65.245 1.00 40.79 C ATOM 798 O LYS A 633 25.233 22.623 65.335 1.00 41.41 O ATOM 799 CB LYS A 633 26.080 25.233 65.479 1.00 40.46 C ATOM 800 CG LYS A 633 26.502 26.549 66.117 1.00 41.28 C ATOM 801 CD LYS A 633 25.437 27.622 65.921 1.00 43.26 C ATOM 802 CE LYS A 633 25.892 28.975 66.458 1.00 43.46 C ATOM 803 NZ LYS A 633 24.948 30.064 66.056 1.00 45.08 N ATOM 804 N ILE A 634 27.258 21.807 64.818 1.00 40.89 N ATOM 805 CA ILE A 634 26.743 20.500 64.431 1.00 41.48 C ATOM 806 C ILE A 634 26.514 19.648 65.678 1.00 42.34 C ATOM 807 O ILE A 634 25.709 18.718 65.677 1.00 42.84 O ATOM 808 CB ILE A 634 27.742 19.775 63.501 1.00 41.22 C ATOM 809 CG1 ILE A 634 27.818 20.510 62.163 1.00 41.52 C ATOM 810 CG2 ILE A 634 27.334 18.320 63.313 1.00 40.18 C ATOM 811 CD1 ILE A 634 28.950 20.044 61.268 1.00 42.19 C ATOM 812 N GLN A 635 27.231 19.981 66.742 1.00 42.86 N ATOM 813 CA GLN A 635 27.145 19.261 68.005 1.00 43.96 C ATOM 814 C GLN A 635 25.717 18.979 68.483 1.00 44.80 C ATOM 815 O GLN A 635 25.414 17.871 68.926 1.00 45.06 O ATOM 816 CB GLN A 635 27.911 20.045 69.070 1.00 44.10 C ATOM 817 CG GLN A 635 27.835 19.486 70.471 1.00 45.22 C ATOM 818 CD GLN A 635 28.758 20.226 71.419 1.00 45.45 C ATOM 819 OE1 GLN A 635 28.811 21.456 71.415 1.00 46.41 O ATOM 820 NE2 GLN A 635 29.488 19.481 72.237 1.00 45.04 N ATOM 821 N ASN A 636 24.842 19.976 68.382 1.00 45.32 N ATOM 822 CA ASN A 636 23.456 19.839 68.829 1.00 45.41 C ATOM 823 C ASN A 636 22.628 18.866 68.001 1.00 44.87 C ATOM 824 O ASN A 636 21.577 18.406 68.443 1.00 44.79 O ATOM 825 CB ASN A 636 22.761 21.206 68.822 1.00 46.54 C ATOM 826 CG ASN A 636 23.479 22.230 69.676 1.00 48.24 C ATOM 827 OD1 ASN A 636 23.661 22.037 70.881 1.00 48.35 O ATOM 828 ND2 ASN A 636 23.896 23.331 69.054 1.00 49.31 N ATOM 829 N LYS A 637 23.095 18.551 66.801 1.00 44.14 N ATOM 830 CA LYS A 637 22.356 17.650 65.932 1.00 43.64 C ATOM 831 C LYS A 637 22.744 16.183 66.074 1.00 42.68 C ATOM 832 O LYS A 637 22.204 15.331 65.370 1.00 42.44 O ATOM 833 CB LYS A 637 22.532 18.087 64.477 1.00 44.85 C ATOM 834 CG LYS A 637 22.133 19.532 64.233 1.00 47.07 C ATOM 835 CD LYS A 637 22.184 19.891 62.759 1.00 48.61 C ATOM 836 CE LYS A 637 21.603 21.274 62.524 1.00 49.01 C ATOM 837 NZ LYS A 637 21.367 21.524 61.079 1.00 50.60 N ATOM 838 N LEU A 638 23.661 15.883 66.990 1.00 41.27 N ATOM 839 CA LEU A 638 24.117 14.508 67.179 1.00 40.07 C ATOM 840 C LEU A 638 24.068 14.083 68.638 1.00 39.23 C ATOM 841 O LEU A 638 23.930 14.916 69.530 1.00 39.72 O ATOM 842 CB LEU A 638 25.552 14.360 66.662 1.00 39.45 C ATOM 843 CG LEU A 638 25.827 14.860 65.244 1.00 39.08 C ATOM 844 CD1 LEU A 638 27.300 14.669 64.902 1.00 38.86 C ATOM 845 CD2 LEU A 638 24.949 14.113 64.266 1.00 38.98 C ATOM 846 N THR A 639 24.186 12.782 68.877 1.00 38.25 N ATOM 847 CA THR A 639 24.165 12.259 70.240 1.00 37.28 C ATOM 848 C THR A 639 25.589 12.195 70.771 1.00 36.75 C ATOM 849 O THR A 639 26.548 12.351 70.011 1.00 36.05 O ATOM 850 CB THR A 639 23.581 10.836 70.302 1.00 37.19 C ATOM 851 OG1 THR A 639 24.478 9.923 69.656 1.00 36.69 O ATOM 852 CG2 THR A 639 22.220 10.781 69.614 1.00 36.32 C ATOM 853 N ASP A 640 25.729 11.961 72.073 1.00 36.26 N ATOM 854 CA ASP A 640 27.052 11.867 72.672 1.00 36.32 C ATOM 855 C ASP A 640 27.850 10.697 72.099 1.00 35.02 C ATOM 856 O ASP A 640 29.059 10.816 71.884 1.00 34.76 O ATOM 857 CB ASP A 640 26.955 11.723 74.193 1.00 38.26 C ATOM 858 CG ASP A 640 26.662 13.039 74.887 1.00 39.96 C ATOM 859 OD1 ASP A 640 26.725 14.087 74.217 1.00 40.78 O ATOM 860 OD2 ASP A 640 26.381 13.027 76.105 1.00 42.06 O ATOM 861 N LEU A 641 27.182 9.571 71.854 1.00 33.08 N ATOM 862 CA LEU A 641 27.868 8.406 71.308 1.00 32.34 C ATOM 863 C LEU A 641 28.357 8.663 69.890 1.00 31.96 C ATOM 864 O LEU A 641 29.424 8.185 69.504 1.00 31.71 O ATOM 865 CB LEU A 641 26.959 7.172 71.332 1.00 31.19 C ATOM 866 CG LEU A 641 26.635 6.602 72.720 1.00 30.69 C ATOM 867 CD1 LEU A 641 25.708 5.397 72.577 1.00 29.71 C ATOM 868 CD2 LEU A 641 27.915 6.201 73.427 1.00 28.60 C ATOM 869 N GLU A 642 27.578 9.418 69.118 1.00 31.24 N ATOM 870 CA GLU A 642 27.955 9.735 67.747 1.00 30.84 C ATOM 871 C GLU A 642 29.158 10.675 67.742 1.00 29.56 C ATOM 872 O GLU A 642 30.075 10.506 66.943 1.00 28.91 O ATOM 873 CB GLU A 642 26.772 10.363 67.003 1.00 31.18 C ATOM 874 CG GLU A 642 25.542 9.454 66.983 1.00 31.88 C ATOM 875 CD GLU A 642 24.388 10.020 66.184 1.00 32.73 C ATOM 876 OE1 GLU A 642 24.076 11.221 66.340 1.00 32.27 O ATOM 877 OE2 GLU A 642 23.786 9.255 65.405 1.00 32.80 O ATOM 878 N ILE A 643 29.153 11.645 68.652 1.00 28.48 N ATOM 879 CA ILE A 643 30.249 12.607 68.771 1.00 27.88 C ATOM 880 C ILE A 643 31.525 11.900 69.224 1.00 26.94 C ATOM 881 O ILE A 643 32.612 12.190 68.728 1.00 25.90 O ATOM 882 CB ILE A 643 29.892 13.731 69.777 1.00 28.36 C ATOM 883 CG1 ILE A 643 28.781 14.610 69.186 1.00 28.88 C ATOM 884 CG2 ILE A 643 31.132 14.562 70.107 1.00 28.38 C ATOM 885 CD1 ILE A 643 28.190 15.609 70.155 1.00 30.17 C ATOM 886 N LEU A 644 31.379 10.966 70.160 1.00 26.15 N ATOM 887 CA LEU A 644 32.507 10.191 70.670 1.00 25.82 C ATOM 888 C LEU A 644 33.118 9.369 69.535 1.00 25.67 C ATOM 889 O LEU A 644 34.334 9.338 69.359 1.00 24.76 O ATOM 890 CB LEU A 644 32.026 9.253 71.783 1.00 27.00 C ATOM 891 CG LEU A 644 32.975 8.165 72.294 1.00 27.02 C ATOM 892 CD1 LEU A 644 34.141 8.798 73.026 1.00 27.74 C ATOM 893 CD2 LEU A 644 32.219 7.227 73.226 1.00 28.47 C ATOM 894 N ALA A 645 32.257 8.700 68.770 1.00 25.19 N ATOM 895 CA ALA A 645 32.691 7.863 67.659 1.00 24.40 C ATOM 896 C ALA A 645 33.376 8.669 66.555 1.00 23.83 C ATOM 897 O ALA A 645 34.407 8.254 66.020 1.00 22.94 O ATOM 898 CB ALA A 645 31.496 7.106 67.088 1.00 24.19 C ATOM 899 N LEU A 646 32.794 9.817 66.218 1.00 22.61 N ATOM 900 CA LEU A 646 33.338 10.689 65.181 1.00 23.41 C ATOM 901 C LEU A 646 34.742 11.182 65.521 1.00 22.68 C ATOM 902 O LEU A 646 35.642 11.141 64.678 1.00 23.43 O ATOM 903 CB LEU A 646 32.423 11.901 64.966 1.00 23.21 C ATOM 904 CG LEU A 646 31.104 11.691 64.214 1.00 24.57 C ATOM 905 CD1 LEU A 646 30.240 12.953 64.327 1.00 24.83 C ATOM 906 CD2 LEU A 646 31.389 11.369 62.759 1.00 23.27 C ATOM 907 N LEU A 647 34.927 11.645 66.754 1.00 21.87 N ATOM 908 CA LEU A 647 36.226 12.159 67.175 1.00 21.96 C ATOM 909 C LEU A 647 37.288 11.077 67.088 1.00 21.42 C ATOM 910 O LEU A 647 38.385 11.311 66.570 1.00 21.70 O ATOM 911 CB LEU A 647 36.159 12.713 68.603 1.00 21.42 C ATOM 912 CG LEU A 647 37.450 13.388 69.094 1.00 20.22 C ATOM 913 CD1 LEU A 647 37.841 14.506 68.160 1.00 20.73 C ATOM 914 CD2 LEU A 647 37.238 13.934 70.497 1.00 21.29 C ATOM 915 N ILE A 648 36.964 9.894 67.602 1.00 21.60 N ATOM 916 CA ILE A 648 37.883 8.760 67.560 1.00 20.41 C ATOM 917 C ILE A 648 38.162 8.363 66.108 1.00 20.06 C ATOM 918 O ILE A 648 39.311 8.114 65.729 1.00 19.99 O ATOM 919 CB ILE A 648 37.299 7.543 68.325 1.00 21.47 C ATOM 920 CG1 ILE A 648 37.280 7.838 69.828 1.00 21.87 C ATOM 921 CG2 ILE A 648 38.137 6.292 68.049 1.00 21.57 C ATOM 922 CD1 ILE A 648 36.453 6.853 70.655 1.00 22.97 C ATOM 923 N ALA A 649 37.110 8.300 65.297 1.00 19.50 N ATOM 924 CA ALA A 649 37.246 7.944 63.880 1.00 19.34 C ATOM 925 C ALA A 649 38.146 8.955 63.157 1.00 19.12 C ATOM 926 O ALA A 649 39.080 8.585 62.439 1.00 20.17 O ATOM 927 CB ALA A 649 35.863 7.911 63.214 1.00 18.41 C ATOM 928 N ALA A 650 37.860 10.236 63.340 1.00 18.82 N ATOM 929 CA ALA A 650 38.666 11.276 62.694 1.00 19.02 C ATOM 930 C ALA A 650 40.144 11.053 63.009 1.00 18.08 C ATOM 931 O ALA A 650 40.977 11.025 62.111 1.00 19.28 O ATOM 932 CB ALA A 650 38.223 12.661 63.175 1.00 18.68 C ATOM 933 N LEU A 651 40.458 10.871 64.287 1.00 18.24 N ATOM 934 CA LEU A 651 41.835 10.659 64.722 1.00 18.02 C ATOM 935 C LEU A 651 42.427 9.332 64.229 1.00 18.25 C ATOM 936 O LEU A 651 43.638 9.228 64.006 1.00 16.91 O ATOM 937 CB LEU A 651 41.909 10.695 66.260 1.00 19.00 C ATOM 938 CG LEU A 651 41.688 12.022 66.998 1.00 19.68 C ATOM 939 CD1 LEU A 651 41.356 11.761 68.457 1.00 19.96 C ATOM 940 CD2 LEU A 651 42.942 12.893 66.883 1.00 20.84 C ATOM 941 N SER A 652 41.575 8.323 64.054 1.00 17.83 N ATOM 942 CA SER A 652 42.040 6.990 63.655 1.00 19.00 C ATOM 943 C SER A 652 41.872 6.594 62.193 1.00 19.23 C ATOM 944 O SER A 652 42.416 5.579 61.773 1.00 19.98 O ATOM 945 CB SER A 652 41.311 5.918 64.483 1.00 19.45 C ATOM 946 OG SER A 652 41.370 6.180 65.871 1.00 21.36 O ATOM 947 N HIS A 653 41.139 7.385 61.421 1.00 19.68 N ATOM 948 CA HIS A 653 40.833 7.008 60.046 1.00 19.43 C ATOM 949 C HIS A 653 41.941 6.658 59.063 1.00 20.17 C ATOM 950 O HIS A 653 41.654 6.011 58.064 1.00 20.39 O ATOM 951 CB HIS A 653 39.887 8.040 59.414 1.00 18.92 C ATOM 952 CG HIS A 653 40.586 9.209 58.789 1.00 18.98 C ATOM 953 ND1 HIS A 653 41.019 10.296 59.519 1.00 19.24 N ATOM 954 CD2 HIS A 653 40.923 9.452 57.503 1.00 17.63 C ATOM 955 CE1 HIS A 653 41.590 11.163 58.700 1.00 17.52 C ATOM 956 NE2 HIS A 653 41.543 10.675 57.472 1.00 17.34 N ATOM 957 N ASP A 654 43.185 7.078 59.301 1.00 19.61 N ATOM 958 CA ASP A 654 44.278 6.740 58.379 1.00 20.03 C ATOM 959 C ASP A 654 45.429 5.997 59.049 1.00 20.31 C ATOM 960 O ASP A 654 46.558 6.012 58.546 1.00 20.95 O ATOM 961 CB ASP A 654 44.834 7.998 57.722 1.00 19.88 C ATOM 962 CG ASP A 654 44.038 8.416 56.506 1.00 20.74 C ATOM 963 OD1 ASP A 654 43.609 7.511 55.752 1.00 21.43 O ATOM 964 OD2 ASP A 654 43.859 9.632 56.255 1.00 20.20 O ATOM 965 N LEU A 655 45.160 5.387 60.200 1.00 19.89 N ATOM 966 CA LEU A 655 46.183 4.669 60.937 1.00 20.36 C ATOM 967 C LEU A 655 47.025 3.704 60.097 1.00 20.84 C ATOM 968 O LEU A 655 46.496 2.945 59.273 1.00 20.21 O ATOM 969 CB LEU A 655 45.550 3.903 62.107 1.00 20.08 C ATOM 970 CG LEU A 655 45.222 4.666 63.390 1.00 21.11 C ATOM 971 CD1 LEU A 655 44.266 3.835 64.250 1.00 20.20 C ATOM 972 CD2 LEU A 655 46.503 4.957 64.148 1.00 21.02 C ATOM 973 N ASP A 656 48.340 3.774 60.302 1.00 21.70 N ATOM 974 CA ASP A 656 49.314 2.917 59.638 1.00 23.38 C ATOM 975 C ASP A 656 49.397 3.104 58.126 1.00 24.19 C ATOM 976 O ASP A 656 49.942 2.262 57.426 1.00 23.92 O ATOM 977 CB ASP A 656 49.005 1.454 59.992 1.00 24.45 C ATOM 978 CG ASP A 656 50.169 0.514 59.698 1.00 25.59 C ATOM 979 OD1 ASP A 656 51.328 0.865 60.015 1.00 25.86 O ATOM 980 OD2 ASP A 656 49.924 −0.588 59.166 1.00 26.82 O ATOM 981 N HIS A 657 48.865 4.215 57.626 1.00 25.15 N ATOM 982 CA HIS A 657 48.905 4.508 56.189 1.00 25.34 C ATOM 983 C HIS A 657 50.365 4.555 55.728 1.00 26.06 C ATOM 984 O HIS A 657 51.219 5.137 56.397 1.00 25.14 O ATOM 985 CB HIS A 657 48.231 5.854 55.921 1.00 24.46 C ATOM 986 CG HIS A 657 47.716 6.012 54.524 1.00 24.00 C ATOM 987 ND1 HIS A 657 48.542 6.066 53.422 1.00 22.91 N ATOM 988 CD2 HIS A 657 46.453 6.146 54.056 1.00 23.03 C ATOM 989 CE1 HIS A 657 47.809 6.230 52.335 1.00 24.09 C ATOM 990 NE2 HIS A 657 46.538 6.281 52.694 1.00 22.84 N ATOM 991 N ARG A 658 50.656 3.947 54.583 1.00 27.49 N ATOM 992 CA ARG A 658 52.026 3.935 54.081 1.00 29.96 C ATOM 993 C ARG A 658 52.238 4.827 52.861 1.00 30.06 C ATOM 994 O ARG A 658 53.321 4.846 52.285 1.00 29.63 O ATOM 995 CB ARG A 658 52.446 2.497 53.758 1.00 32.14 C ATOM 996 CG ARG A 658 52.573 1.591 54.989 1.00 35.42 C ATOM 997 CD ARG A 658 52.639 0.118 54.582 1.00 39.58 C ATOM 998 NE ARG A 658 53.007 −0.760 55.694 1.00 42.66 N ATOM 999 CZ ARG A 658 54.221 −0.807 56.235 1.00 43.93 C ATOM 1000 NH1 ARG A 658 55.190 −0.027 55.767 1.00 45.35 N ATOM 1001 NH2 ARG A 658 54.472 −1.632 57.241 1.00 44.87 N ATOM 1002 N GLY A 659 51.210 5.573 52.474 1.00 30.34 N ATOM 1003 CA GLY A 659 51.335 6.442 51.318 1.00 31.78 C ATOM 1004 C GLY A 659 50.508 5.932 50.155 1.00 32.86 C ATOM 1005 O GLY A 659 50.460 4.726 49.903 1.00 32.41 O ATOM 1006 N VAL A 660 49.854 6.844 49.444 1.00 34.47 N ATOM 1007 CA VAL A 660 49.015 6.460 48.315 1.00 37.07 C ATOM 1008 C VAL A 660 49.716 5.555 47.301 1.00 38.55 C ATOM 1009 O VAL A 660 49.075 4.704 46.687 1.00 39.62 O ATOM 1010 CB VAL A 660 48.467 7.697 47.570 1.00 37.04 C ATOM 1011 CG1 VAL A 660 47.547 8.489 48.483 1.00 37.23 C ATOM 1012 CG2 VAL A 660 49.622 8.568 47.084 1.00 37.36 C ATOM 1013 N ASN A 661 51.024 5.726 47.131 1.00 40.07 N ATOM 1014 CA ASN A 661 51.766 4.908 46.171 1.00 42.63 C ATOM 1015 C ASN A 661 52.388 3.644 46.746 1.00 44.42 C ATOM 1016 O ASN A 661 53.207 2.991 46.092 1.00 44.01 O ATOM 1017 CB ASN A 661 52.849 5.735 45.481 1.00 41.27 C ATOM 1018 CG ASN A 661 52.270 6.828 44.615 1.00 41.09 C ATOM 1019 OD1 ASN A 661 51.226 6.644 43.993 1.00 40.35 O ATOM 1020 ND2 ASN A 661 52.947 7.970 44.560 1.00 40.87 N ATOM 1021 N ASN A 662 52.004 3.300 47.968 1.00 46.67 N ATOM 1022 CA ASN A 662 52.520 2.099 48.596 1.00 49.44 C ATOM 1023 C ASN A 662 51.453 1.007 48.483 1.00 51.35 C ATOM 1024 O ASN A 662 50.285 1.227 48.808 1.00 50.63 O ATOM 1025 CB ASN A 662 52.877 2.380 50.055 1.00 50.07 C ATOM 1026 CG ASN A 662 53.501 1.186 50.736 1.00 50.69 C ATOM 1027 OD1 ASN A 662 52.844 0.165 50.939 1.00 50.93 O ATOM 1028 ND2 ASN A 662 54.779 1.302 51.089 1.00 50.91 N ATOM 1029 N SER A 663 51.872 −0.162 48.004 1.00 54.13 N ATOM 1030 CA SER A 663 50.990 −1.311 47.792 1.00 57.21 C ATOM 1031 C SER A 663 51.130 −2.385 48.872 1.00 59.09 C ATOM 1032 O SER A 663 50.423 −3.394 48.846 1.00 59.12 O ATOM 1033 CB SER A 663 51.299 −1.949 46.430 1.00 57.27 C ATOM 1034 OG SER A 663 51.323 −0.992 45.384 1.00 58.78 O ATOM 1035 N TYR A 664 52.038 −2.171 49.816 1.00 61.54 N ATOM 1036 CA TYR A 664 52.280 −3.143 50.874 1.00 64.28 C ATOM 1037 C TYR A 664 51.029 −3.686 51.565 1.00 65.88 C ATOM 1038 O TYR A 664 50.698 −4.863 51.420 1.00 66.03 O ATOM 1039 CB TYR A 664 53.224 −2.552 51.918 1.00 64.85 C ATOM 1040 CG TYR A 664 53.771 −3.590 52.855 1.00 65.69 C ATOM 1041 CD1 TYR A 664 53.082 −3.952 54.012 1.00 66.10 C ATOM 1042 CD2 TYR A 664 54.963 −4.246 52.562 1.00 66.20 C ATOM 1043 CE1 TYR A 664 53.573 −4.948 54.855 1.00 66.66 C ATOM 1044 CE2 TYR A 664 55.460 −5.238 53.389 1.00 66.43 C ATOM 1045 CZ TYR A 664 54.765 −5.586 54.535 1.00 66.77 C ATOM 1046 OH TYR A 664 55.277 −6.563 55.357 1.00 66.80 O ATOM 1047 N ILE A 665 50.336 −2.834 52.312 1.00 67.73 N ATOM 1048 CA ILE A 665 49.137 −3.260 53.033 1.00 69.76 C ATOM 1049 C ILE A 665 48.178 −4.107 52.206 1.00 71.22 C ATOM 1050 O ILE A 665 47.630 −5.094 52.696 1.00 71.40 O ATOM 1051 CB ILE A 665 48.342 −2.058 53.581 1.00 69.70 C ATOM 1052 CG1 ILE A 665 49.233 −1.230 54.507 1.00 69.76 C ATOM 1053 CG2 ILE A 665 47.104 −2.553 54.334 1.00 69.55 C ATOM 1054 CD1 ILE A 665 48.601 0.060 54.979 1.00 69.31 C ATOM 1055 N GLN A 666 47.976 −3.720 50.954 1.00 72.97 N ATOM 1056 CA GLN A 666 47.063 −4.436 50.075 1.00 74.96 C ATOM 1057 C GLN A 666 47.620 −5.773 49.582 1.00 76.03 C ATOM 1058 O GLN A 666 46.895 −6.764 49.510 1.00 76.08 O ATOM 1059 CB GLN A 666 46.711 −3.540 48.889 1.00 75.43 C ATOM 1060 CG GLN A 666 46.253 −2.154 49.314 1.00 76.40 C ATOM 1061 CD GLN A 666 46.411 −1.128 48.215 1.00 77.09 C ATOM 1062 OE1 GLN A 666 45.698 −1.156 47.211 1.00 77.79 O ATOM 1063 NE2 GLN A 666 47.362 −0.216 48.395 1.00 77.18 N ATOM 1064 N ARG A 667 48.907 −5.799 49.251 1.00 77.41 N ATOM 1065 CA ARG A 667 49.549 −7.015 48.761 1.00 78.95 C ATOM 1066 C ARG A 667 49.920 −7.987 49.877 1.00 79.77 C ATOM 1067 O ARG A 667 50.309 −9.124 49.613 1.00 79.73 O ATOM 1068 CB ARG A 667 50.797 −6.654 47.947 1.00 79.33 C ATOM 1069 CG ARG A 667 50.479 −6.078 46.575 1.00 79.99 C ATOM 1070 CD ARG A 667 51.651 −5.305 45.989 1.00 80.54 C ATOM 1071 NE ARG A 667 51.369 −4.805 44.642 1.00 81.26 N ATOM 1072 CZ ARG A 667 50.286 −4.109 44.302 1.00 81.68 C ATOM 1073 NH1 ARG A 667 49.359 −3.820 45.207 1.00 81.97 N ATOM 1074 NH2 ARG A 667 50.127 −3.696 43.052 1.00 81.83 N ATOM 1075 N SER A 668 49.800 −7.543 51.124 1.00 80.94 N ATOM 1076 CA SER A 668 50.121 −8.402 52.256 1.00 82.25 C ATOM 1077 C SER A 668 48.904 −9.235 52.651 1.00 83.15 C ATOM 1078 O SER A 668 48.996 −10.114 53.509 1.00 83.20 O ATOM 1079 CB SER A 668 50.599 −7.570 53.451 1.00 82.24 C ATOM 1080 OG SER A 668 49.583 −6.700 53.915 1.00 82.67 O ATOM 1081 N GLU A 669 47.765 −8.953 52.022 1.00 84.18 N ATOM 1082 CA GLU A 669 46.535 −9.689 52.291 1.00 85.37 C ATOM 1083 C GLU A 669 46.329 −10.706 51.162 1.00 85.83 C ATOM 1084 O GLU A 669 47.111 −10.746 50.217 1.00 85.97 O ATOM 1085 CB GLU A 669 45.344 −8.721 52.379 1.00 85.95 C ATOM 1086 CG GLU A 669 44.101 −9.308 53.047 1.00 86.88 C ATOM 1087 CD GLU A 669 43.094 −8.245 53.468 1.00 87.30 C ATOM 1088 OE1 GLU A 669 42.505 −7.581 52.588 1.00 87.69 O ATOM 1089 OE2 GLU A 669 42.893 −8.071 54.690 1.00 87.62 O ATOM 1090 N HIS A 670 45.275 −11.509 51.272 1.00 86.43 N ATOM 1091 CA HIS A 670 44.924 −12.570 50.315 1.00 86.94 C ATOM 1092 C HIS A 670 44.807 −12.183 48.825 1.00 87.14 C ATOM 1093 O HIS A 670 43.702 −12.129 48.278 1.00 87.16 O ATOM 1094 CB HIS A 670 43.617 −13.217 50.784 1.00 87.12 C ATOM 1095 CG HIS A 670 43.594 −14.707 50.655 1.00 87.37 C ATOM 1096 ND1 HIS A 670 43.201 −15.354 49.503 1.00 87.40 N ATOM 1097 CD2 HIS A 670 43.940 −15.679 51.533 1.00 87.48 C ATOM 1098 CE1 HIS A 670 43.307 −16.658 49.676 1.00 87.52 C ATOM 1099 NE2 HIS A 670 43.753 −16.883 50.898 1.00 87.57 N ATOM 1100 N PRO A 671 45.954 −11.992 48.141 1.00 87.32 N ATOM 1101 CA PRO A 671 46.158 −11.621 46.733 1.00 87.30 C ATOM 1102 C PRO A 671 44.974 −10.913 46.064 1.00 87.21 C ATOM 1103 O PRO A 671 44.912 −9.678 46.034 1.00 87.39 O ATOM 1104 CB PRO A 671 46.543 −12.953 46.124 1.00 87.35 C ATOM 1105 CG PRO A 671 47.608 −13.414 47.203 1.00 87.49 C ATOM 1106 CD PRO A 671 47.052 −12.889 48.558 1.00 87.38 C ATOM 1107 N LEU A 672 44.040 −11.689 45.526 1.00 87.03 N ATOM 1108 CA LEU A 672 42.849 −11.111 44.916 1.00 86.85 C ATOM 1109 C LEU A 672 41.986 −10.568 46.058 1.00 86.61 C ATOM 1110 O LEU A 672 40.759 −10.500 45.964 1.00 86.38 O ATOM 1111 CB LEU A 672 42.074 −12.173 44.134 1.00 86.85 C ATOM 1112 CG LEU A 672 42.616 −12.629 42.778 1.00 86.78 C ATOM 1113 CD1 LEU A 672 41.449 −13.223 42.016 1.00 86.88 C ATOM 1114 CD2 LEU A 672 43.209 −11.467 41.980 1.00 86.69 C ATOM 1115 N ALA A 673 42.666 −10.184 47.134 1.00 86.38 N ATOM 1116 CA ALA A 673 42.056 −9.649 48.342 1.00 85.97 C ATOM 1117 C ALA A 673 41.396 −8.294 48.112 1.00 85.67 C ATOM 1118 O ALA A 673 40.617 −7.836 48.946 1.00 85.70 O ATOM 1119 CB ALA A 673 43.120 −9.536 49.440 1.00 85.94 C ATOM 1120 N GLN A 674 41.703 −7.660 46.984 1.00 85.15 N ATOM 1121 CA GLN A 674 41.135 −6.351 46.661 1.00 84.67 C ATOM 1122 C GLN A 674 39.613 −6.349 46.446 1.00 83.76 C ATOM 1123 O GLN A 674 38.920 −5.432 46.892 1.00 83.70 O ATOM 1124 CB GLN A 674 41.820 −5.767 45.409 1.00 85.59 C ATOM 1125 CG GLN A 674 43.283 −5.362 45.596 1.00 86.53 C ATOM 1126 CD GLN A 674 43.928 −4.836 44.318 1.00 87.08 C ATOM 1127 OE1 GLN A 674 44.249 −5.603 43.409 1.00 87.49 O ATOM 1128 NE2 GLN A 674 44.117 −3.520 44.246 1.00 87.54 N ATOM 1129 N LEU A 675 39.087 −7.379 45.791 1.00 82.61 N ATOM 1130 CA LEU A 675 37.659 −7.418 45.488 1.00 81.28 C ATOM 1131 C LEU A 675 36.731 −8.045 46.530 1.00 80.23 C ATOM 1132 O LEU A 675 35.535 −7.748 46.559 1.00 80.12 O ATOM 1133 CB LEU A 675 37.476 −8.074 44.121 1.00 81.47 C ATOM 1134 CG LEU A 675 38.479 −7.486 43.117 1.00 81.42 C ATOM 1135 CD1 LEU A 675 38.302 −8.152 41.783 1.00 81.50 C ATOM 1136 CD2 LEU A 675 38.300 −5.976 42.991 1.00 81.40 C ATOM 1137 N TYR A 676 37.278 −8.902 47.387 1.00 78.95 N ATOM 1138 CA TYR A 676 36.504 −9.539 48.449 1.00 77.57 C ATOM 1139 C TYR A 676 36.194 −8.482 49.503 1.00 75.67 C ATOM 1140 O TYR A 676 35.105 −7.894 49.509 1.00 75.94 O ATOM 1141 CB TYR A 676 37.312 −10.705 49.026 1.00 79.09 C ATOM 1142 CG TYR A 676 37.402 −11.814 48.013 1.00 80.54 C ATOM 1143 CD1 TYR A 676 36.295 −12.616 47.761 1.00 81.23 C ATOM 1144 CD2 TYR A 676 38.525 −11.963 47.197 1.00 81.05 C ATOM 1145 CE1 TYR A 676 36.285 −13.513 46.717 1.00 82.14 C ATOM 1146 CE2 TYR A 676 38.528 −12.871 46.138 1.00 81.83 C ATOM 1147 CZ TYR A 676 37.398 −13.641 45.905 1.00 82.47 C ATOM 1148 OH TYR A 676 37.351 −14.519 44.847 1.00 83.12 O ATOM 1149 N CYS A 677 37.156 −8.242 50.388 1.00 72.77 N ATOM 1150 CA CYS A 677 37.015 −7.217 51.416 1.00 69.74 C ATOM 1151 C CYS A 677 37.345 −5.878 50.748 1.00 67.28 C ATOM 1152 O CYS A 677 38.502 −5.461 50.740 1.00 67.18 O ATOM 1153 CB CYS A 677 38.004 −7.471 52.554 1.00 69.91 C ATOM 1154 SG CYS A 677 38.281 −6.031 53.600 1.00 69.81 S ATOM 1155 N HIS A 678 36.343 −5.206 50.185 1.00 64.08 N ATOM 1156 CA HIS A 678 36.585 −3.929 49.514 1.00 60.63 C ATOM 1157 C HIS A 678 36.845 −2.795 50.514 1.00 57.06 C ATOM 1158 O HIS A 678 36.557 −2.937 51.704 1.00 56.42 O ATOM 1159 CB HIS A 678 35.418 −3.583 48.584 1.00 62.54 C ATOM 1160 CG HIS A 678 35.740 −2.513 47.586 1.00 64.43 C ATOM 1161 ND1 HIS A 678 35.900 −1.191 47.941 1.00 65.61 N ATOM 1162 CD2 HIS A 678 35.957 −2.573 46.250 1.00 65.04 C ATOM 1163 CE1 HIS A 678 36.200 −0.481 46.868 1.00 65.84 C ATOM 1164 NE2 HIS A 678 36.242 −1.296 45.828 1.00 66.00 N ATOM 1165 N SER A 679 37.375 −1.672 50.024 1.00 52.71 N ATOM 1166 CA SER A 679 37.738 −0.546 50.886 1.00 48.04 C ATOM 1167 C SER A 679 38.777 −1.164 51.807 1.00 45.31 C ATOM 1168 O SER A 679 38.749 −0.987 53.024 1.00 44.54 O ATOM 1169 CB SER A 679 36.543 −0.054 51.710 1.00 47.68 C ATOM 1170 OG SER A 679 35.600 0.620 50.902 1.00 46.88 O ATOM 1171 N ILE A 680 39.692 −1.908 51.195 1.00 41.93 N ATOM 1172 CA ILE A 680 40.732 −2.618 51.917 1.00 39.16 C ATOM 1173 C ILE A 680 41.550 −1.767 52.888 1.00 36.30 C ATOM 1174 O ILE A 680 41.776 −2.177 54.017 1.00 34.33 O ATOM 1175 CB ILE A 680 41.667 −3.345 50.923 1.00 39.71 C ATOM 1176 CG1 ILE A 680 42.631 −4.257 51.682 1.00 39.57 C ATOM 1177 CG2 ILE A 680 42.410 −2.331 50.072 1.00 40.14 C ATOM 1178 CD1 ILE A 680 43.426 −5.178 50.777 1.00 41.19 C ATOM 1179 N MET A 681 41.989 −0.591 52.453 1.00 34.80 N ATOM 1180 CA MET A 681 42.769 0.291 53.315 1.00 33.59 C ATOM 1181 C MET A 681 41.961 0.715 54.538 1.00 31.42 C ATOM 1182 O MET A 681 42.434 0.617 55.666 1.00 32.21 O ATOM 1183 CB MET A 681 43.218 1.538 52.545 1.00 34.53 C ATOM 1184 CG MET A 681 44.359 1.299 51.576 1.00 37.45 C ATOM 1185 SD MET A 681 45.861 0.792 52.439 1.00 40.06 S ATOM 1186 CE MET A 681 46.370 2.359 53.165 1.00 39.76 C ATOM 1187 N GLU A 682 40.738 1.178 54.312 1.00 29.63 N ATOM 1188 CA GLU A 682 39.886 1.620 55.409 1.00 27.83 C ATOM 1189 C GLU A 682 39.612 0.519 56.436 1.00 27.23 C ATOM 1190 O GLU A 682 39.533 0.796 57.634 1.00 25.93 O ATOM 1191 CB GLU A 682 38.571 2.183 54.867 1.00 27.59 C ATOM 1192 CG GLU A 682 38.720 3.476 54.048 1.00 28.02 C ATOM 1193 CD GLU A 682 39.481 3.279 52.739 1.00 29.28 C ATOM 1194 OE1 GLU A 682 39.379 2.186 52.145 1.00 27.32 O ATOM 1195 OE2 GLU A 682 40.169 4.224 52.294 1.00 28.60 O ATOM 1196 N HIS A 683 39.469 −0.726 55.981 1.00 26.34 N ATOM 1197 CA HIS A 683 39.233 −1.826 56.914 1.00 25.90 C ATOM 1198 C HIS A 683 40.477 −1.990 57.769 1.00 25.31 C ATOM 1199 O HIS A 683 40.389 −2.259 58.968 1.00 25.62 O ATOM 1200 CB HIS A 683 38.930 −3.129 56.165 1.00 26.85 C ATOM 1201 CG HIS A 683 37.492 −3.274 55.775 1.00 26.92 C ATOM 1202 ND1 HIS A 683 36.504 −3.580 56.685 1.00 26.84 N ATOM 1203 CD2 HIS A 683 36.868 −3.103 54.586 1.00 26.72 C ATOM 1204 CE1 HIS A 683 35.332 −3.588 56.075 1.00 27.20 C ATOM 1205 NE2 HIS A 683 35.526 −3.301 54.800 1.00 27.36 N ATOM 1206 N HIS A 684 41.634 −1.815 57.142 1.00 24.66 N ATOM 1207 CA HIS A 684 42.908 −1.919 57.839 1.00 24.73 C ATOM 1208 C HIS A 684 42.977 −0.803 58.880 1.00 24.18 C ATOM 1209 O HIS A 684 43.317 −1.045 60.032 1.00 23.63 O ATOM 1210 CB HIS A 684 44.068 −1.786 56.843 1.00 25.27 C ATOM 1211 CG HIS A 684 45.424 −1.777 57.484 1.00 26.02 C ATOM 1212 ND1 HIS A 684 45.952 −2.873 58.134 1.00 25.96 N ATOM 1213 CD2 HIS A 684 46.347 −0.792 57.600 1.00 25.92 C ATOM 1214 CE1 HIS A 684 47.138 −2.563 58.625 1.00 26.23 C ATOM 1215 NE2 HIS A 684 47.401 −1.305 58.316 1.00 26.02 N ATOM 1216 N HIS A 685 42.645 0.423 58.476 1.00 24.00 N ATOM 1217 CA HIS A 685 42.688 1.539 59.416 1.00 22.79 C ATOM 1218 C HIS A 685 41.791 1.247 60.615 1.00 22.55 C ATOM 1219 O HIS A 685 42.163 1.510 61.758 1.00 21.67 O ATOM 1220 CB HIS A 685 42.245 2.847 58.747 1.00 21.96 C ATOM 1221 CG HIS A 685 43.073 3.228 57.561 1.00 21.33 C ATOM 1222 ND1 HIS A 685 44.451 3.190 57.570 1.00 21.52 N ATOM 1223 CD2 HIS A 685 42.718 3.676 56.332 1.00 21.14 C ATOM 1224 CE1 HIS A 685 44.910 3.597 56.399 1.00 20.01 C ATOM 1225 NE2 HIS A 685 43.878 3.898 55.631 1.00 20.93 N ATOM 1226 N PHE A 686 40.607 0.707 60.356 1.00 23.14 N ATOM 1227 CA PHE A 686 39.703 0.384 61.446 1.00 24.54 C ATOM 1228 C PHE A 686 40.293 −0.691 62.359 1.00 25.13 C ATOM 1229 O PHE A 686 40.228 −0.575 63.585 1.00 25.48 O ATOM 1230 CB PHE A 686 38.353 −0.103 60.934 1.00 25.17 C ATOM 1231 CG PHE A 686 37.445 −0.552 62.032 1.00 26.21 C ATOM 1232 CD1 PHE A 686 36.893 0.376 62.913 1.00 26.80 C ATOM 1233 CD2 PHE A 686 37.220 −1.902 62.253 1.00 27.27 C ATOM 1234 CE1 PHE A 686 36.134 −0.033 63.998 1.00 27.27 C ATOM 1235 CE2 PHE A 686 36.458 −2.326 63.345 1.00 27.89 C ATOM 1236 CZ PHE A 686 35.918 −1.390 64.217 1.00 27.93 C ATOM 1237 N ASP A 687 40.858 −1.739 61.768 1.00 25.65 N ATOM 1238 CA ASP A 687 41.454 −2.802 62.566 1.00 26.17 C ATOM 1239 C ASP A 687 42.517 −2.207 63.481 1.00 24.82 C ATOM 1240 O ASP A 687 42.629 −2.585 64.642 1.00 22.98 O ATOM 1241 CB ASP A 687 42.085 −3.880 61.677 1.00 29.27 C ATOM 1242 CG ASP A 687 41.052 −4.792 61.046 1.00 33.31 C ATOM 1243 OD1 ASP A 687 40.054 −5.123 61.729 1.00 36.16 O ATOM 1244 OD2 ASP A 687 41.240 −5.193 59.874 1.00 35.83 O ATOM 1245 N GLN A 688 43.295 −1.272 62.946 1.00 24.28 N ATOM 1246 CA GLN A 688 44.340 −0.610 63.724 1.00 24.28 C ATOM 1247 C GLN A 688 43.691 0.160 64.871 1.00 22.95 C ATOM 1248 O GLN A 688 44.133 0.081 66.015 1.00 23.59 O ATOM 1249 CB GLN A 688 45.135 0.347 62.831 1.00 24.61 C ATOM 1250 CG GLN A 688 46.039 −0.360 61.836 1.00 27.56 C ATOM 1251 CD GLN A 688 47.204 −1.066 62.509 1.00 29.57 C ATOM 1252 OE1 GLN A 688 48.056 −0.427 63.126 1.00 29.65 O ATOM 1253 NE2 GLN A 688 47.246 −2.391 62.394 1.00 29.57 N ATOM 1254 N CYS A 689 42.632 0.896 64.551 1.00 22.05 N ATOM 1255 CA CYS A 689 41.897 1.674 65.540 1.00 20.99 C ATOM 1256 C CYS A 689 41.416 0.758 66.667 1.00 21.59 C ATOM 1257 O CYS A 689 41.630 1.047 67.844 1.00 20.81 O ATOM 1258 CB CYS A 689 40.696 2.350 64.875 1.00 20.11 C ATOM 1259 SG CYS A 689 39.584 3.219 65.998 1.00 22.22 S ATOM 1260 N LEU A 690 40.782 −0.353 66.292 1.00 21.89 N ATOM 1261 CA LEU A 690 40.256 −1.308 67.261 1.00 23.02 C ATOM 1262 C LEU A 690 41.340 −1.925 68.150 1.00 23.78 C ATOM 1263 O LEU A 690 41.143 −2.073 69.359 1.00 23.96 O ATOM 1264 CB LEU A 690 39.483 −2.421 66.538 1.00 23.05 C ATOM 1265 CG LEU A 690 38.736 −3.419 67.428 1.00 24.22 C ATOM 1266 CD1 LEU A 690 37.698 −2.680 68.262 1.00 25.51 C ATOM 1267 CD2 LEU A 690 38.069 −4.484 66.568 1.00 23.71 C ATOM 1268 N MET A 691 42.476 −2.291 67.561 1.00 24.50 N ATOM 1269 CA MET A 691 43.561 −2.887 68.339 1.00 25.97 C ATOM 1270 C MET A 691 44.051 −1.904 69.408 1.00 25.62 C ATOM 1271 O MET A 691 44.337 −2.290 70.540 1.00 24.15 O ATOM 1272 CB MET A 691 44.731 −3.279 67.432 1.00 29.30 C ATOM 1273 CG MET A 691 44.359 −4.233 66.302 1.00 35.33 C ATOM 1274 SD MET A 691 45.786 −4.814 65.321 1.00 42.12 S ATOM 1275 CE MET A 691 45.364 −6.545 65.127 1.00 39.80 C ATOM 1276 N ILE A 692 44.158 −0.631 69.042 1.00 24.96 N ATOM 1277 CA ILE A 692 44.602 0.375 69.992 1.00 24.69 C ATOM 1278 C ILE A 692 43.562 0.536 71.097 1.00 24.93 C ATOM 1279 O ILE A 692 43.910 0.640 72.276 1.00 25.24 O ATOM 1280 CB ILE A 692 44.848 1.739 69.297 1.00 25.05 C ATOM 1281 CG1 ILE A 692 46.037 1.615 68.335 1.00 24.23 C ATOM 1282 CG2 ILE A 692 45.133 2.815 70.346 1.00 24.86 C ATOM 1283 CD1 ILE A 692 46.287 2.869 67.477 1.00 25.03 C ATOM 1284 N LEU A 693 42.287 0.539 70.715 1.00 24.89 N ATOM 1285 CA LEU A 693 41.191 0.670 71.677 1.00 25.65 C ATOM 1286 C LEU A 693 41.132 −0.478 72.680 1.00 25.72 C ATOM 1287 O LEU A 693 40.566 −0.325 73.760 1.00 26.68 O ATOM 1288 CB LEU A 693 39.843 0.745 70.952 1.00 24.67 C ATOM 1289 CG LEU A 693 39.479 2.080 70.301 1.00 24.93 C ATOM 1290 CD1 LEU A 693 38.165 1.929 69.516 1.00 21.93 C ATOM 1291 CD2 LEU A 693 39.365 3.153 71.387 1.00 23.04 C ATOM 1292 N ASN A 694 41.700 −1.625 72.317 1.00 26.40 N ATOM 1293 CA ASN A 694 41.693 −2.792 73.194 1.00 27.39 C ATOM 1294 C ASN A 694 43.034 −3.051 73.873 1.00 27.89 C ATOM 1295 O ASN A 694 43.151 −3.975 74.671 1.00 28.05 O ATOM 1296 CB ASN A 694 41.272 −4.050 72.421 1.00 28.45 C ATOM 1297 CG ASN A 694 39.790 −4.065 72.095 1.00 30.82 C ATOM 1298 OD1 ASN A 694 38.957 −3.787 72.955 1.00 34.31 O ATOM 1299 ND2 ASN A 694 39.454 −4.393 70.856 1.00 31.56 N ATOM 1300 N SER A 695 44.041 −2.241 73.556 1.00 27.21 N ATOM 1301 CA SER A 695 45.363 −2.397 74.152 1.00 27.57 C ATOM 1302 C SER A 695 45.307 −2.062 75.637 1.00 27.38 C ATOM 1303 O SER A 695 44.589 −1.157 76.049 1.00 26.73 O ATOM 1304 CB SER A 695 46.376 −1.468 73.471 1.00 28.21 C ATOM 1305 OG SER A 695 46.624 −1.866 72.138 1.00 30.28 O ATOM 1306 N PRO A 696 46.065 −2.793 76.464 1.00 28.12 N ATOM 1307 CA PRO A 696 46.041 −2.500 77.898 1.00 27.96 C ATOM 1308 C PRO A 696 46.466 −1.062 78.197 1.00 27.75 C ATOM 1309 O PRO A 696 47.410 −0.538 77.591 1.00 26.30 O ATOM 1310 CB PRO A 696 46.997 −3.540 78.488 1.00 29.31 C ATOM 1311 CG PRO A 696 47.902 −3.884 77.353 1.00 30.80 C ATOM 1312 CD PRO A 696 46.967 −3.918 76.167 1.00 29.71 C ATOM 1313 N GLY A 697 45.741 −0.429 79.115 1.00 26.84 N ATOM 1314 CA GLY A 697 46.035 0.939 79.497 1.00 26.60 C ATOM 1315 C GLY A 697 45.593 1.991 78.495 1.00 26.28 C ATOM 1316 O GLY A 697 45.784 3.187 78.735 1.00 25.78 O ATOM 1317 N ASN A 698 44.996 1.556 77.387 1.00 25.57 N ATOM 1318 CA ASN A 698 44.532 2.462 76.334 1.00 25.23 C ATOM 1319 C ASN A 698 43.022 2.423 76.119 1.00 25.16 C ATOM 1320 O ASN A 698 42.506 3.074 75.207 1.00 23.86 O ATOM 1321 CB ASN A 698 45.196 2.101 75.003 1.00 25.93 C ATOM 1322 CG ASN A 698 46.678 2.402 74.983 1.00 26.87 C ATOM 1323 OD1 ASN A 698 47.109 3.406 74.423 1.00 27.03 O ATOM 1324 ND2 ASN A 698 47.468 1.530 75.596 1.00 27.99 N ATOM 1325 N GLN A 699 42.307 1.676 76.952 1.00 24.20 N ATOM 1326 CA GLN A 699 40.866 1.531 76.769 1.00 24.34 C ATOM 1327 C GLN A 699 39.974 2.697 77.176 1.00 24.77 C ATOM 1328 O GLN A 699 39.279 2.637 78.199 1.00 24.40 O ATOM 1329 CB GLN A 699 40.419 0.241 77.453 1.00 24.44 C ATOM 1330 CG GLN A 699 41.034 −0.982 76.796 1.00 25.18 C ATOM 1331 CD GLN A 699 41.109 −2.174 77.716 1.00 27.03 C ATOM 1332 OE1 GLN A 699 40.108 −2.590 78.293 1.00 27.20 O ATOM 1333 NE2 GLN A 699 42.306 −2.731 77.861 1.00 27.83 N ATOM 1334 N ILE A 700 39.964 3.746 76.350 1.00 24.07 N ATOM 1335 CA ILE A 700 39.158 4.924 76.645 1.00 24.94 C ATOM 1336 C ILE A 700 37.656 4.679 76.571 1.00 25.08 C ATOM 1337 O ILE A 700 36.880 5.540 76.956 1.00 24.59 O ATOM 1338 CB ILE A 700 39.504 6.125 75.718 1.00 25.34 C ATOM 1339 CG1 ILE A 700 39.326 5.732 74.248 1.00 24.79 C ATOM 1340 CG2 ILE A 700 40.926 6.605 75.998 1.00 25.16 C ATOM 1341 CD1 ILE A 700 39.398 6.922 73.294 1.00 25.40 C ATOM 1342 N LEU A 701 37.244 3.514 76.078 1.00 25.70 N ATOM 1343 CA LEU A 701 35.821 3.193 76.011 1.00 25.97 C ATOM 1344 C LEU A 701 35.387 2.188 77.101 1.00 27.21 C ATOM 1345 O LEU A 701 34.256 1.692 77.093 1.00 26.42 O ATOM 1346 CB LEU A 701 35.472 2.646 74.625 1.00 25.82 C ATOM 1347 CG LEU A 701 35.635 3.626 73.458 1.00 25.35 C ATOM 1348 CD1 LEU A 701 35.220 2.952 72.170 1.00 26.35 C ATOM 1349 CD2 LEU A 701 34.786 4.867 73.696 1.00 26.20 C ATOM 1350 N SER A 702 36.277 1.901 78.046 1.00 28.19 N ATOM 1351 CA SER A 702 35.958 0.948 79.109 1.00 29.27 C ATOM 1352 C SER A 702 34.778 1.417 79.958 1.00 30.38 C ATOM 1353 O SER A 702 34.119 0.611 80.612 1.00 30.51 O ATOM 1354 CB SER A 702 37.175 0.724 80.013 1.00 28.94 C ATOM 1355 OG SER A 702 37.434 1.859 80.828 1.00 28.49 O ATOM 1356 N GLY A 703 34.516 2.722 79.943 1.00 30.20 N ATOM 1357 CA GLY A 703 33.427 3.271 80.728 1.00 30.06 C ATOM 1358 C GLY A 703 32.039 3.030 80.165 1.00 30.10 C ATOM 1359 O GLY A 703 31.047 3.122 80.893 1.00 30.40 O ATOM 1360 N LEU A 704 31.958 2.729 78.873 1.00 28.97 N ATOM 1361 CA LEU A 704 30.675 2.482 78.228 1.00 28.63 C ATOM 1362 C LEU A 704 30.137 1.102 78.589 1.00 29.50 C ATOM 1363 O LEU A 704 30.897 0.205 78.935 1.00 28.98 O ATOM 1364 CB LEU A 704 30.820 2.565 76.703 1.00 27.31 C ATOM 1365 CG LEU A 704 30.928 3.920 75.991 1.00 27.53 C ATOM 1366 CD1 LEU A 704 32.024 4.768 76.610 1.00 25.43 C ATOM 1367 CD2 LEU A 704 31.202 3.678 74.512 1.00 25.52 C ATOM 1368 N SER A 705 28.822 0.937 78.510 1.00 30.52 N ATOM 1369 CA SER A 705 28.214 −0.363 78.783 1.00 31.09 C ATOM 1370 C SER A 705 28.422 −1.158 77.498 1.00 31.45 C ATOM 1371 O SER A 705 28.741 −0.578 76.458 1.00 30.33 O ATOM 1372 CB SER A 705 26.717 −0.213 79.054 1.00 30.76 C ATOM 1373 OG SER A 705 26.054 0.320 77.920 1.00 30.72 O ATOM 1374 N ILE A 706 28.246 −2.474 77.561 1.00 31.49 N ATOM 1375 CA ILE A 706 28.431 −3.303 76.379 1.00 31.58 C ATOM 1376 C ILE A 706 27.570 −2.841 75.206 1.00 31.39 C ATOM 1377 O ILE A 706 28.012 −2.872 74.053 1.00 31.47 O ATOM 1378 CB ILE A 706 28.135 −4.801 76.681 1.00 32.51 C ATOM 1379 CG1 ILE A 706 28.449 −5.650 75.447 1.00 32.96 C ATOM 1380 CG2 ILE A 706 26.673 −4.990 77.069 1.00 31.23 C ATOM 1381 CD1 ILE A 706 29.853 −5.469 74.922 1.00 34.07 C ATOM 1382 N GLU A 707 26.348 −2.400 75.484 1.00 31.64 N ATOM 1383 CA GLU A 707 25.476 −1.944 74.405 1.00 32.73 C ATOM 1384 C GLU A 707 25.951 −0.609 73.833 1.00 32.15 C ATOM 1385 O GLU A 707 25.903 −0.391 72.623 1.00 31.17 O ATOM 1386 CB GLU A 707 24.029 −1.826 74.892 1.00 34.95 C ATOM 1387 CG GLU A 707 23.401 −3.164 75.291 1.00 38.64 C ATOM 1388 CD GLU A 707 23.431 −4.194 74.164 1.00 41.39 C ATOM 1389 OE1 GLU A 707 22.881 −3.910 73.074 1.00 41.69 O ATOM 1390 OE2 GLU A 707 24.003 −5.292 74.373 1.00 43.16 O ATOM 1391 N GLU A 708 26.406 0.287 74.703 1.00 31.80 N ATOM 1392 CA GLU A 708 26.906 1.582 74.253 1.00 31.35 C ATOM 1393 C GLU A 708 28.138 1.339 73.385 1.00 30.77 C ATOM 1394 O GLU A 708 28.323 1.970 72.341 1.00 29.85 O ATOM 1395 CB GLU A 708 27.285 2.448 75.453 1.00 31.93 C ATOM 1396 CG GLU A 708 26.091 3.064 76.174 1.00 34.14 C ATOM 1397 CD GLU A 708 26.489 3.793 77.441 1.00 35.02 C ATOM 1398 OE1 GLU A 708 25.709 4.654 77.900 1.00 37.29 O ATOM 1399 OE2 GLU A 708 27.573 3.501 77.987 1.00 34.33 O ATOM 1400 N TYR A 709 28.963 0.397 73.829 1.00 29.79 N ATOM 1401 CA TYR A 709 30.194 0.042 73.142 1.00 29.26 C ATOM 1402 C TYR A 709 29.956 −0.492 71.728 1.00 29.67 C ATOM 1403 O TYR A 709 30.575 −0.024 70.771 1.00 29.38 O ATOM 1404 CB TYR A 709 30.955 −0.992 73.972 1.00 27.77 C ATOM 1405 CG TYR A 709 32.288 −1.396 73.390 1.00 27.67 C ATOM 1406 CD1 TYR A 709 33.355 −0.492 73.327 1.00 26.72 C ATOM 1407 CD2 TYR A 709 32.486 −2.685 72.898 1.00 26.83 C ATOM 1408 CE1 TYR A 709 34.587 −0.871 72.785 1.00 26.23 C ATOM 1409 CE2 TYR A 709 33.705 −3.074 72.357 1.00 26.97 C ATOM 1410 CZ TYR A 709 34.750 −2.168 72.302 1.00 26.95 C ATOM 1411 OH TYR A 709 35.947 −2.572 71.758 1.00 26.33 O ATOM 1412 N LYS A 710 29.067 −1.471 71.590 1.00 29.83 N ATOM 1413 CA LYS A 710 28.794 −2.033 70.275 1.00 30.53 C ATOM 1414 C LYS A 710 28.268 −0.963 69.311 1.00 30.43 C ATOM 1415 O LYS A 710 28.594 −0.968 68.123 1.00 29.85 O ATOM 1416 CB LYS A 710 27.793 −3.186 70.388 1.00 31.48 C ATOM 1417 CG LYS A 710 28.287 −4.342 71.264 1.00 33.54 C ATOM 1418 CD LYS A 710 27.442 −5.608 71.092 1.00 34.58 C ATOM 1419 CE LYS A 710 25.971 −5.366 71.399 1.00 36.04 C ATOM 1420 NZ LYS A 710 25.156 −6.623 71.310 1.00 37.04 N ATOM 1421 N THR A 711 27.456 −0.048 69.826 1.00 29.80 N ATOM 1422 CA THR A 711 26.905 1.021 69.003 1.00 29.96 C ATOM 1423 C THR A 711 28.018 1.978 68.568 1.00 29.26 C ATOM 1424 O THR A 711 28.100 2.364 67.398 1.00 29.58 O ATOM 1425 CB THR A 711 25.822 1.808 69.774 1.00 30.83 C ATOM 1426 OG1 THR A 711 24.729 0.932 70.087 1.00 31.30 O ATOM 1427 CG2 THR A 711 25.307 2.972 68.936 1.00 31.90 C ATOM 1428 N THR A 712 28.881 2.346 69.507 1.00 28.32 N ATOM 1429 CA THR A 712 29.976 3.254 69.195 1.00 27.69 C ATOM 1430 C THR A 712 30.919 2.610 68.184 1.00 27.19 C ATOM 1431 O THR A 712 31.237 3.220 67.167 1.00 26.50 O ATOM 1432 CB THR A 712 30.738 3.658 70.469 1.00 27.67 C ATOM 1433 OG1 THR A 712 29.834 4.319 71.368 1.00 28.26 O ATOM 1434 CG2 THR A 712 31.882 4.615 70.129 1.00 26.58 C ATOM 1435 N LEU A 713 31.351 1.375 68.444 1.00 26.56 N ATOM 1436 CA LEU A 713 32.234 0.679 67.506 1.00 26.61 C ATOM 1437 C LEU A 713 31.649 0.662 66.100 1.00 26.37 C ATOM 1438 O LEU A 713 32.368 0.857 65.128 1.00 26.97 O ATOM 1439 CB LEU A 713 32.485 −0.772 67.931 1.00 26.07 C ATOM 1440 CG LEU A 713 33.630 −1.105 68.881 1.00 27.61 C ATOM 1441 CD1 LEU A 713 33.852 −2.629 68.863 1.00 27.54 C ATOM 1442 CD2 LEU A 713 34.906 −0.382 68.447 1.00 27.62 C ATOM 1443 N LYS A 714 30.347 0.415 65.998 1.00 27.41 N ATOM 1444 CA LYS A 714 29.667 0.370 64.703 1.00 28.02 C ATOM 1445 C LYS A 714 29.737 1.716 63.988 1.00 27.75 C ATOM 1446 O LYS A 714 30.006 1.776 62.788 1.00 27.18 O ATOM 1447 CB LYS A 714 28.201 −0.033 64.887 1.00 30.89 C ATOM 1448 CG LYS A 714 27.346 0.108 63.624 1.00 33.65 C ATOM 1449 CD LYS A 714 25.906 −0.340 63.874 1.00 35.22 C ATOM 1450 CE LYS A 714 25.070 −0.245 62.608 1.00 36.34 C ATOM 1451 NZ LYS A 714 23.723 −0.861 62.793 1.00 38.97 N ATOM 1452 N ILE A 715 29.475 2.792 64.727 1.00 26.60 N ATOM 1453 CA ILE A 715 29.529 4.127 64.152 1.00 25.95 C ATOM 1454 C ILE A 715 30.964 4.457 63.748 1.00 25.38 C ATOM 1455 O ILE A 715 31.192 5.057 62.697 1.00 25.14 O ATOM 1456 CB ILE A 715 29.014 5.185 65.152 1.00 26.71 C ATOM 1457 CG1 ILE A 715 27.520 4.953 65.430 1.00 26.93 C ATOM 1458 CG2 ILE A 715 29.237 6.582 64.592 1.00 26.26 C ATOM 1459 CD1 ILE A 715 26.961 5.780 66.588 1.00 27.42 C ATOM 1460 N ILE A 716 31.925 4.054 64.577 1.00 24.12 N ATOM 1461 CA ILE A 716 33.338 4.309 64.288 1.00 24.37 C ATOM 1462 C ILE A 716 33.757 3.588 63.021 1.00 24.11 C ATOM 1463 O ILE A 716 34.477 4.138 62.192 1.00 24.32 O ATOM 1464 CB ILE A 716 34.266 3.804 65.412 1.00 22.91 C ATOM 1465 CG1 ILE A 716 34.063 4.627 66.681 1.00 22.79 C ATOM 1466 CG2 ILE A 716 35.725 3.859 64.945 1.00 21.65 C ATOM 1467 CD1 ILE A 716 34.854 4.109 67.872 1.00 21.67 C ATOM 1468 N LYS A 717 33.325 2.338 62.887 1.00 24.40 N ATOM 1469 CA LYS A 717 33.672 1.550 61.713 1.00 25.22 C ATOM 1470 C LYS A 717 33.091 2.172 60.445 1.00 25.70 C ATOM 1471 O LYS A 717 33.791 2.312 59.445 1.00 25.83 O ATOM 1472 CB LYS A 717 33.166 0.116 61.870 1.00 25.53 C ATOM 1473 CG LYS A 717 33.576 −0.814 60.750 1.00 26.18 C ATOM 1474 CD LYS A 717 33.242 −2.257 61.120 1.00 27.27 C ATOM 1475 CE LYS A 717 33.568 −3.222 60.001 1.00 26.85 C ATOM 1476 NZ LYS A 717 33.342 −4.627 60.436 1.00 27.16 N ATOM 1477 N GLN A 718 31.812 2.540 60.488 1.00 25.97 N ATOM 1478 CA GLN A 718 31.157 3.156 59.337 1.00 27.02 C ATOM 1479 C GLN A 718 31.836 4.495 59.029 1.00 26.67 C ATOM 1480 O GLN A 718 32.057 4.847 57.869 1.00 26.29 O ATOM 1481 CB GLN A 718 29.666 3.390 59.632 1.00 28.70 C ATOM 1482 CG GLN A 718 28.898 2.112 60.008 1.00 32.94 C ATOM 1483 CD GLN A 718 27.441 2.374 60.380 1.00 35.85 C ATOM 1484 OE1 GLN A 718 27.144 3.163 61.285 1.00 37.30 O ATOM 1485 NE2 GLN A 718 26.525 1.704 59.684 1.00 37.21 N ATOM 1486 N ALA A 719 32.168 5.234 60.082 1.00 25.30 N ATOM 1487 CA ALA A 719 32.811 6.532 59.918 1.00 25.10 C ATOM 1488 C ALA A 719 34.138 6.380 59.182 1.00 24.15 C ATOM 1489 O ALA A 719 34.433 7.137 58.263 1.00 25.50 O ATOM 1490 CB ALA A 719 33.032 7.186 61.287 1.00 24.05 C ATOM 1491 N ILE A 720 34.935 5.396 59.582 1.00 23.09 N ATOM 1492 CA ILE A 720 36.219 5.176 58.942 1.00 22.38 C ATOM 1493 C ILE A 720 36.061 4.641 57.519 1.00 22.35 C ATOM 1494 O ILE A 720 36.779 5.065 56.617 1.00 22.16 O ATOM 1495 CB ILE A 720 37.114 4.232 59.810 1.00 21.78 C ATOM 1496 CG1 ILE A 720 37.439 4.936 61.136 1.00 22.04 C ATOM 1497 CG2 ILE A 720 38.388 3.862 59.067 1.00 20.92 C ATOM 1498 CD1 ILE A 720 38.437 4.223 62.031 1.00 21.45 C ATOM 1499 N LEU A 721 35.118 3.723 57.303 1.00 22.70 N ATOM 1500 CA LEU A 721 34.913 3.186 55.959 1.00 22.52 C ATOM 1501 C LEU A 721 34.489 4.317 55.037 1.00 23.02 C ATOM 1502 O LEU A 721 34.854 4.344 53.866 1.00 24.19 O ATOM 1503 CB LEU A 721 33.843 2.079 55.960 1.00 23.25 C ATOM 1504 CG LEU A 721 34.195 0.786 56.709 1.00 24.15 C ATOM 1505 CD1 LEU A 721 33.127 −0.289 56.430 1.00 24.76 C ATOM 1506 CD2 LEU A 721 35.552 0.292 56.256 1.00 22.86 C ATOM 1507 N ALA A 722 33.729 5.260 55.581 1.00 23.48 N ATOM 1508 CA ALA A 722 33.253 6.406 54.815 1.00 23.24 C ATOM 1509 C ALA A 722 34.389 7.249 54.236 1.00 23.40 C ATOM 1510 O ALA A 722 34.203 7.946 53.234 1.00 22.99 O ATOM 1511 CB ALA A 722 32.364 7.276 55.694 1.00 23.42 C ATOM 1512 N THR A 723 35.564 7.198 54.855 1.00 23.00 N ATOM 1513 CA THR A 723 36.681 7.994 54.357 1.00 23.00 C ATOM 1514 C THR A 723 37.261 7.439 53.058 1.00 23.73 C ATOM 1515 O THR A 723 38.224 7.984 52.517 1.00 22.86 O ATOM 1516 CB THR A 723 37.787 8.145 55.424 1.00 22.33 C ATOM 1517 OG1 THR A 723 38.344 6.864 55.749 1.00 21.17 O ATOM 1518 CG2 THR A 723 37.195 8.773 56.683 1.00 23.62 C ATOM 1519 N ASP A 724 36.673 6.348 52.569 1.00 24.32 N ATOM 1520 CA ASP A 724 37.084 5.760 51.297 1.00 25.97 C ATOM 1521 C ASP A 724 36.369 6.636 50.270 1.00 26.42 C ATOM 1522 O ASP A 724 35.144 6.594 50.171 1.00 26.39 O ATOM 1523 CB ASP A 724 36.580 4.315 51.177 1.00 27.17 C ATOM 1524 CG ASP A 724 36.796 3.725 49.787 1.00 27.66 C ATOM 1525 OD1 ASP A 724 37.278 4.440 48.890 1.00 28.01 O ATOM 1526 OD2 ASP A 724 36.478 2.535 49.587 1.00 29.76 O ATOM 1527 N LEU A 725 37.117 7.438 49.521 1.00 27.06 N ATOM 1528 CA LEU A 725 36.487 8.319 48.537 1.00 29.11 C ATOM 1529 C LEU A 725 35.582 7.571 47.561 1.00 29.87 C ATOM 1530 O LEU A 725 34.623 8.138 47.042 1.00 30.24 O ATOM 1531 CB LEU A 725 37.545 9.114 47.771 1.00 29.36 C ATOM 1532 CG LEU A 725 38.197 10.261 48.549 1.00 28.95 C ATOM 1533 CD1 LEU A 725 39.389 10.798 47.777 1.00 29.96 C ATOM 1534 CD2 LEU A 725 37.169 11.356 48.795 1.00 29.37 C ATOM 1535 N ALA A 726 35.881 6.300 47.313 1.00 30.76 N ATOM 1536 CA ALA A 726 35.048 5.513 46.409 1.00 31.28 C ATOM 1537 C ALA A 726 33.642 5.442 46.997 1.00 31.31 C ATOM 1538 O ALA A 726 32.652 5.522 46.270 1.00 32.05 O ATOM 1539 CB ALA A 726 35.625 4.113 46.236 1.00 31.66 C ATOM 1540 N LEU A 727 33.548 5.311 48.318 1.00 31.27 N ATOM 1541 CA LEU A 727 32.244 5.246 48.961 1.00 30.98 C ATOM 1542 C LEU A 727 31.576 6.616 48.991 1.00 31.58 C ATOM 1543 O LEU A 727 30.349 6.720 48.899 1.00 31.00 O ATOM 1544 CB LEU A 727 32.358 4.687 50.382 1.00 30.98 C ATOM 1545 CG LEU A 727 32.819 3.234 50.537 1.00 31.73 C ATOM 1546 CD1 LEU A 727 32.570 2.784 51.981 1.00 32.31 C ATOM 1547 CD2 LEU A 727 32.058 2.323 49.570 1.00 32.38 C ATOM 1548 N TYR A 728 32.381 7.667 49.124 1.00 31.57 N ATOM 1549 CA TYR A 728 31.849 9.027 49.138 1.00 32.42 C ATOM 1550 C TYR A 728 31.173 9.333 47.798 1.00 33.23 C ATOM 1551 O TYR A 728 30.047 9.823 47.754 1.00 33.29 O ATOM 1552 CB TYR A 728 32.972 10.041 49.383 1.00 31.75 C ATOM 1553 CG TYR A 728 32.617 11.453 48.960 1.00 31.60 C ATOM 1554 CD1 TYR A 728 31.603 12.159 49.602 1.00 31.22 C ATOM 1555 CD2 TYR A 728 33.265 12.063 47.886 1.00 31.32 C ATOM 1556 CE1 TYR A 728 31.235 13.433 49.185 1.00 31.91 C ATOM 1557 CE2 TYR A 728 32.905 13.340 47.459 1.00 32.61 C ATOM 1558 CZ TYR A 728 31.886 14.017 48.115 1.00 32.46 C ATOM 1559 OH TYR A 728 31.513 15.272 47.700 1.00 34.08 O ATOM 1560 N ILE A 729 31.876 9.048 46.710 1.00 34.82 N ATOM 1561 CA ILE A 729 31.348 9.296 45.374 1.00 37.06 C ATOM 1562 C ILE A 729 30.113 8.451 45.083 1.00 38.48 C ATOM 1563 O ILE A 729 29.237 8.851 44.320 1.00 38.62 O ATOM 1564 CB ILE A 729 32.423 9.025 44.306 1.00 37.19 C ATOM 1565 CG1 ILE A 729 33.463 10.148 44.342 1.00 37.45 C ATOM 1566 CG2 ILE A 729 31.784 8.908 42.926 1.00 38.11 C ATOM 1567 CD1 ILE A 729 34.567 10.002 43.325 1.00 39.16 C ATOM 1568 N LYS A 730 30.040 7.288 45.714 1.00 40.06 N ATOM 1569 CA LYS A 730 28.918 6.381 45.518 1.00 41.63 C ATOM 1570 C LYS A 730 27.660 6.838 46.249 1.00 41.67 C ATOM 1571 O LYS A 730 26.557 6.685 45.735 1.00 42.26 O ATOM 1572 CB LYS A 730 29.310 4.978 45.989 1.00 42.71 C ATOM 1573 CG LYS A 730 28.238 3.912 45.819 1.00 45.29 C ATOM 1574 CD LYS A 730 28.723 2.583 46.401 1.00 46.43 C ATOM 1575 CE LYS A 730 27.695 1.470 46.235 1.00 47.25 C ATOM 1576 NZ LYS A 730 28.195 0.178 46.795 1.00 47.44 N ATOM 1577 N ARG A 731 27.820 7.421 47.433 1.00 41.53 N ATOM 1578 CA ARG A 731 26.665 7.850 48.219 1.00 41.43 C ATOM 1579 C ARG A 731 26.349 9.342 48.247 1.00 41.16 C ATOM 1580 O ARG A 731 25.269 9.733 48.690 1.00 40.19 O ATOM 1581 CB ARG A 731 26.816 7.363 49.662 1.00 42.53 C ATOM 1582 CG ARG A 731 26.993 5.858 49.796 1.00 44.52 C ATOM 1583 CD ARG A 731 27.032 5.454 51.260 1.00 46.07 C ATOM 1584 NE ARG A 731 25.791 5.793 51.950 1.00 47.44 N ATOM 1585 CZ ARG A 731 25.610 5.673 53.262 1.00 47.85 C ATOM 1586 NH1 ARG A 731 26.593 5.219 54.030 1.00 48.63 N ATOM 1587 NH2 ARG A 731 24.449 6.009 53.807 1.00 48.65 N ATOM 1588 N ARG A 732 27.274 10.178 47.787 1.00 41.06 N ATOM 1589 CA ARG A 732 27.041 11.619 47.810 1.00 41.36 C ATOM 1590 C ARG A 732 25.812 12.035 47.003 1.00 41.66 C ATOM 1591 O ARG A 732 25.085 12.953 47.391 1.00 40.90 O ATOM 1592 CB ARG A 732 28.274 12.371 47.300 1.00 40.99 C ATOM 1593 CG ARG A 732 28.540 12.254 45.816 1.00 41.09 C ATOM 1594 CD ARG A 732 29.749 13.096 45.458 1.00 41.63 C ATOM 1595 NE ARG A 732 30.009 13.148 44.023 1.00 41.54 N ATOM 1596 CZ ARG A 732 30.923 13.941 43.470 1.00 41.70 C ATOM 1597 NH1 ARG A 732 31.651 14.738 44.237 1.00 40.94 N ATOM 1598 NH2 ARG A 732 31.106 13.943 42.155 1.00 42.34 N ATOM 1599 N GLY A 733 25.581 11.353 45.886 1.00 41.99 N ATOM 1600 CA GLY A 733 24.436 11.674 45.054 1.00 43.20 C ATOM 1601 C GLY A 733 23.132 11.684 45.829 1.00 43.42 C ATOM 1602 O GLY A 733 22.310 12.582 45.663 1.00 43.61 O ATOM 1603 N GLU A 734 22.940 10.684 46.683 1.00 43.75 N ATOM 1604 CA GLU A 734 21.722 10.586 47.479 1.00 43.80 C ATOM 1605 C GLU A 734 21.604 11.764 48.439 1.00 43.62 C ATOM 1606 O GLU A 734 20.520 12.321 48.620 1.00 43.36 O ATOM 1607 CB GLU A 734 21.714 9.282 48.278 1.00 44.36 C ATOM 1608 CG GLU A 734 20.369 8.969 48.915 1.00 45.73 C ATOM 1609 CD GLU A 734 20.459 7.910 49.995 1.00 46.03 C ATOM 1610 OE1 GLU A 734 21.223 6.936 49.821 1.00 46.17 O ATOM 1611 OE2 GLU A 734 19.754 8.051 51.016 1.00 47.16 O ATOM 1612 N PHE A 735 22.726 12.125 49.059 1.00 43.39 N ATOM 1613 CA PHE A 735 22.785 13.237 50.010 1.00 43.06 C ATOM 1614 C PHE A 735 22.430 14.542 49.305 1.00 43.37 C ATOM 1615 O PHE A 735 21.651 15.345 49.816 1.00 42.91 O ATOM 1616 CB PHE A 735 24.197 13.321 50.618 1.00 42.33 C ATOM 1617 CG PHE A 735 24.370 14.412 51.647 1.00 41.76 C ATOM 1618 CD1 PHE A 735 23.483 14.539 52.708 1.00 41.33 C ATOM 1619 CD2 PHE A 735 25.447 15.293 51.569 1.00 41.57 C ATOM 1620 CE1 PHE A 735 23.666 15.525 53.678 1.00 41.65 C ATOM 1621 CE2 PHE A 735 25.639 16.280 52.533 1.00 40.91 C ATOM 1622 CZ PHE A 735 24.749 16.397 53.590 1.00 40.90 C ATOM 1623 N PHE A 736 23.005 14.739 48.124 1.00 44.18 N ATOM 1624 CA PHE A 736 22.755 15.935 47.335 1.00 45.75 C ATOM 1625 C PHE A 736 21.302 15.948 46.871 1.00 47.41 C ATOM 1626 O PHE A 736 20.627 16.975 46.941 1.00 47.33 O ATOM 1627 CB PHE A 736 23.692 15.958 46.125 1.00 45.63 C ATOM 1628 CG PHE A 736 25.150 16.063 46.488 1.00 44.55 C ATOM 1629 CD1 PHE A 736 26.131 15.644 45.597 1.00 44.33 C ATOM 1630 CD2 PHE A 736 25.541 16.605 47.710 1.00 44.32 C ATOM 1631 CE1 PHE A 736 27.484 15.759 45.915 1.00 44.21 C ATOM 1632 CE2 PHE A 736 26.891 16.726 48.039 1.00 44.19 C ATOM 1633 CZ PHE A 736 27.863 16.303 47.138 1.00 44.12 C ATOM 1634 N GLU A 737 20.829 14.793 46.413 1.00 48.92 N ATOM 1635 CA GLU A 737 19.459 14.645 45.934 1.00 51.07 C ATOM 1636 C GLU A 737 18.456 14.976 47.038 1.00 51.84 C ATOM 1637 O GLU A 737 17.473 15.683 46.807 1.00 51.75 O ATOM 1638 CB GLU A 737 19.232 13.211 45.449 1.00 51.73 C ATOM 1639 CG GLU A 737 17.941 13.001 44.678 1.00 53.05 C ATOM 1640 CD GLU A 737 17.632 11.533 44.464 1.00 53.37 C ATOM 1641 OE1 GLU A 737 18.546 10.783 44.058 1.00 53.50 O ATOM 1642 OE2 GLU A 737 16.474 11.129 44.697 1.00 54.22 O ATOM 1643 N LEU A 738 18.709 14.460 48.238 1.00 53.21 N ATOM 1644 CA LEU A 738 17.830 14.697 49.378 1.00 54.70 C ATOM 1645 C LEU A 738 17.785 16.170 49.764 1.00 55.83 C ATOM 1646 O LEU A 738 16.793 16.644 50.312 1.00 56.02 O ATOM 1647 CB LEU A 738 18.286 13.868 50.584 1.00 54.83 C ATOM 1648 CG LEU A 738 18.146 12.344 50.484 1.00 55.40 C ATOM 1649 CD1 LEU A 738 18.818 11.684 51.683 1.00 55.13 C ATOM 1650 CD2 LEU A 738 16.673 11.967 50.422 1.00 55.07 C ATOM 1651 N ILE A 739 18.859 16.895 49.476 1.00 57.19 N ATOM 1652 CA ILE A 739 18.913 18.311 49.816 1.00 58.82 C ATOM 1653 C ILE A 739 18.197 19.188 48.793 1.00 59.61 C ATOM 1654 O ILE A 739 17.574 20.182 49.161 1.00 59.57 O ATOM 1655 CB ILE A 739 20.372 18.789 49.978 1.00 58.96 C ATOM 1656 CG1 ILE A 739 21.044 18.005 51.109 1.00 59.35 C ATOM 1657 CG2 ILE A 739 20.404 20.282 50.289 1.00 59.06 C ATOM 1658 CD1 ILE A 739 22.496 18.371 51.344 1.00 59.59 C ATOM 1659 N ARG A 740 18.280 18.823 47.515 1.00 60.85 N ATOM 1660 CA ARG A 740 17.616 19.596 46.466 1.00 62.31 C ATOM 1661 C ARG A 740 16.124 19.642 46.767 1.00 62.97 C ATOM 1662 O ARG A 740 15.529 20.712 46.893 1.00 63.07 O ATOM 1663 CB ARG A 740 17.777 18.936 45.094 1.00 62.90 C ATOM 1664 CG ARG A 740 19.155 18.431 44.744 1.00 63.79 C ATOM 1665 CD ARG A 740 19.056 17.559 43.501 1.00 64.14 C ATOM 1666 NE ARG A 740 20.250 16.750 43.290 1.00 64.41 N ATOM 1667 CZ ARG A 740 20.304 15.704 42.472 1.00 64.58 C ATOM 1668 NH1 ARG A 740 19.228 15.338 41.786 1.00 64.12 N ATOM 1669 NH2 ARG A 740 21.431 15.018 42.348 1.00 64.86 N ATOM 1670 N LYS A 741 15.538 18.453 46.874 1.00 63.77 N ATOM 1671 CA LYS A 741 14.112 18.275 47.130 1.00 64.50 C ATOM 1672 C LYS A 741 13.686 18.654 48.543 1.00 64.84 C ATOM 1673 O LYS A 741 12.519 18.502 48.903 1.00 64.95 O ATOM 1674 CB LYS A 741 13.726 16.818 46.854 1.00 64.52 C ATOM 1675 CG LYS A 741 14.141 16.323 45.473 1.00 64.82 C ATOM 1676 CD LYS A 741 13.930 14.824 45.330 1.00 65.17 C ATOM 1677 CE LYS A 741 14.418 14.321 43.980 1.00 65.79 C ATOM 1678 NZ LYS A 741 14.302 12.837 43.858 1.00 65.91 N ATOM 1679 N ASN A 742 14.628 19.143 49.342 1.00 65.28 N ATOM 1680 CA ASN A 742 14.334 19.540 50.717 1.00 65.79 C ATOM 1681 C ASN A 742 13.693 18.401 51.502 1.00 65.61 C ATOM 1682 O ASN A 742 12.707 18.608 52.210 1.00 65.80 O ATOM 1683 CB ASN A 742 13.390 20.744 50.734 1.00 66.71 C ATOM 1684 CG ASN A 742 13.937 21.930 49.969 1.00 67.73 C ATOM 1685 OD1 ASN A 742 13.301 22.981 49.903 1.00 68.51 O ATOM 1686 ND2 ASN A 742 15.119 21.768 49.383 1.00 68.31 N ATOM 1687 N GLN A 743 14.255 17.202 51.380 1.00 65.23 N ATOM 1688 CA GLN A 743 13.723 16.040 52.082 1.00 64.72 C ATOM 1689 C GLN A 743 14.699 15.506 53.126 1.00 64.19 C ATOM 1690 O GLN A 743 14.475 14.445 53.706 1.00 64.01 O ATOM 1691 CB GLN A 743 13.394 14.927 51.086 1.00 65.26 C ATOM 1692 CG GLN A 743 12.493 15.361 49.947 1.00 66.07 C ATOM 1693 CD GLN A 743 12.051 14.197 49.089 1.00 66.52 C ATOM 1694 OE1 GLN A 743 11.312 13.325 49.542 1.00 67.13 O ATOM 1695 NE2 GLN A 743 12.504 14.173 47.843 1.00 66.88 N ATOM 1696 N PHE A 744 15.781 16.240 53.362 1.00 63.55 N ATOM 1697 CA PHE A 744 16.777 15.815 54.340 1.00 62.76 C ATOM 1698 C PHE A 744 16.184 15.823 55.746 1.00 62.15 C ATOM 1699 O PHE A 744 15.602 16.816 56.178 1.00 62.08 O ATOM 1700 CB PHE A 744 18.003 16.732 54.286 1.00 62.47 C ATOM 1701 CG PHE A 744 19.163 16.237 55.105 1.00 61.73 C ATOM 1702 CD1 PHE A 744 19.779 15.027 54.799 1.00 61.32 C ATOM 1703 CD2 PHE A 744 19.638 16.977 56.182 1.00 61.30 C ATOM 1704 CE1 PHE A 744 20.851 14.562 55.554 1.00 61.16 C ATOM 1705 CE2 PHE A 744 20.709 16.521 56.944 1.00 61.11 C ATOM 1706 CZ PHE A 744 21.316 15.311 56.628 1.00 61.06 C ATOM 1707 N ASN A 745 16.340 14.705 56.450 1.00 61.64 N ATOM 1708 CA ASN A 745 15.820 14.554 57.806 1.00 61.08 C ATOM 1709 C ASN A 745 16.813 13.754 58.649 1.00 60.61 C ATOM 1710 O ASN A 745 17.014 12.562 58.416 1.00 60.41 O ATOM 1711 CB ASN A 745 14.471 13.827 57.762 1.00 61.23 C ATOM 1712 CG ASN A 745 13.856 13.634 59.140 1.00 61.42 C ATOM 1713 OD1 ASN A 745 12.857 12.930 59.284 1.00 61.89 O ATOM 1714 ND2 ASN A 745 14.441 14.263 60.154 1.00 61.19 N ATOM 1715 N LEU A 746 17.431 14.413 59.623 1.00 60.11 N ATOM 1716 CA LEU A 746 18.404 13.757 60.490 1.00 59.73 C ATOM 1717 C LEU A 746 17.748 12.868 61.532 1.00 59.63 C ATOM 1718 O LEU A 746 18.430 12.128 62.242 1.00 59.52 O ATOM 1719 CB LEU A 746 19.273 14.797 61.200 1.00 59.50 C ATOM 1720 CG LEU A 746 20.359 15.491 60.377 1.00 59.37 C ATOM 1721 CD1 LEU A 746 21.074 16.515 61.243 1.00 59.01 C ATOM 1722 CD2 LEU A 746 21.341 14.454 59.852 1.00 59.19 C ATOM 1723 N GLU A 747 16.425 12.937 61.625 1.00 59.72 N ATOM 1724 CA GLU A 747 15.707 12.138 62.606 1.00 59.66 C ATOM 1725 C GLU A 747 15.665 10.662 62.230 1.00 59.07 C ATOM 1726 O GLU A 747 15.595 9.800 63.107 1.00 59.19 O ATOM 1727 CB GLU A 747 14.285 12.672 62.791 1.00 60.71 C ATOM 1728 CG GLU A 747 13.560 12.068 63.987 1.00 62.29 C ATOM 1729 CD GLU A 747 12.212 12.714 64.248 1.00 63.13 C ATOM 1730 OE1 GLU A 747 12.172 13.948 64.453 1.00 63.74 O ATOM 1731 OE2 GLU A 747 11.194 11.988 64.251 1.00 63.51 O ATOM 1732 N ASP A 748 15.708 10.359 60.937 1.00 58.42 N ATOM 1733 CA ASP A 748 15.685 8.962 60.521 1.00 58.07 C ATOM 1734 C ASP A 748 17.118 8.444 60.407 1.00 57.21 C ATOM 1735 O ASP A 748 17.982 9.090 59.813 1.00 57.21 O ATOM 1736 CB ASP A 748 14.941 8.796 59.191 1.00 58.46 C ATOM 1737 CG ASP A 748 15.845 8.938 57.993 1.00 59.25 C ATOM 1738 OD1 ASP A 748 16.432 10.026 57.821 1.00 60.50 O ATOM 1739 OD2 ASP A 748 15.968 7.958 57.224 1.00 58.99 O ATOM 1740 N PRO A 749 17.383 7.262 60.980 1.00 56.22 N ATOM 1741 CA PRO A 749 18.696 6.610 60.983 1.00 55.20 C ATOM 1742 C PRO A 749 19.442 6.530 59.652 1.00 54.35 C ATOM 1743 O PRO A 749 20.660 6.705 59.616 1.00 54.19 O ATOM 1744 CB PRO A 749 18.392 5.227 61.562 1.00 55.53 C ATOM 1745 CG PRO A 749 16.966 4.998 61.164 1.00 55.80 C ATOM 1746 CD PRO A 749 16.348 6.335 61.468 1.00 55.99 C ATOM 1747 N HIS A 750 18.730 6.263 58.562 1.00 53.15 N ATOM 1748 CA HIS A 750 19.396 6.160 57.269 1.00 52.03 C ATOM 1749 C HIS A 750 20.139 7.435 56.894 1.00 51.09 C ATOM 1750 O HIS A 750 21.318 7.396 56.538 1.00 50.70 O ATOM 1751 CB HIS A 750 18.400 5.836 56.156 1.00 51.99 C ATOM 1752 CG HIS A 750 19.023 5.800 54.795 1.00 52.21 C ATOM 1753 ND1 HIS A 750 19.925 4.829 54.416 1.00 52.27 N ATOM 1754 CD2 HIS A 750 18.913 6.643 53.740 1.00 52.07 C ATOM 1755 CE1 HIS A 750 20.344 5.075 53.187 1.00 51.95 C ATOM 1756 NE2 HIS A 750 19.746 6.171 52.755 1.00 51.32 N ATOM 1757 N GLN A 751 19.441 8.563 56.966 1.00 49.77 N ATOM 1758 CA GLN A 751 20.033 9.839 56.610 1.00 48.68 C ATOM 1759 C GLN A 751 21.048 10.326 57.636 1.00 47.88 C ATOM 1760 O GLN A 751 21.986 11.044 57.287 1.00 47.27 O ATOM 1761 CB GLN A 751 18.934 10.884 56.385 1.00 49.16 C ATOM 1762 CG GLN A 751 18.065 10.577 55.164 1.00 49.50 C ATOM 1763 CD GLN A 751 17.060 11.672 54.853 1.00 50.36 C ATOM 1764 OE1 GLN A 751 17.425 12.831 54.651 1.00 50.58 O ATOM 1765 NE2 GLN A 751 15.784 11.306 54.804 1.00 50.55 N ATOM 1766 N LYS A 752 20.872 9.943 58.897 1.00 46.53 N ATOM 1767 CA LYS A 752 21.830 10.352 59.912 1.00 45.81 C ATOM 1768 C LYS A 752 23.161 9.676 59.605 1.00 45.05 C ATOM 1769 O LYS A 752 24.215 10.306 59.658 1.00 44.62 O ATOM 1770 CB LYS A 752 21.363 9.964 61.315 1.00 45.78 C ATOM 1771 CG LYS A 752 22.471 10.084 62.358 1.00 46.32 C ATOM 1772 CD LYS A 752 21.947 10.449 63.739 1.00 47.16 C ATOM 1773 CE LYS A 752 21.585 11.924 63.832 1.00 46.71 C ATOM 1774 NZ LYS A 752 21.360 12.335 65.248 1.00 46.77 N ATOM 1775 N GLU A 753 23.103 8.389 59.276 1.00 44.15 N ATOM 1776 CA GLU A 753 24.307 7.640 58.944 1.00 43.53 C ATOM 1777 C GLU A 753 24.951 8.228 57.691 1.00 41.84 C ATOM 1778 O GLU A 753 26.175 8.333 57.599 1.00 41.15 O ATOM 1779 CB GLU A 753 23.972 6.171 58.687 1.00 45.13 C ATOM 1780 CG GLU A 753 25.189 5.337 58.331 1.00 47.89 C ATOM 1781 CD GLU A 753 24.826 4.013 57.692 1.00 50.08 C ATOM 1782 OE1 GLU A 753 24.329 4.016 56.543 1.00 51.71 O ATOM 1783 OE2 GLU A 753 25.033 2.969 58.339 1.00 51.01 O ATOM 1784 N LEU A 754 24.116 8.599 56.722 1.00 40.36 N ATOM 1785 CA LEU A 754 24.606 9.180 55.478 1.00 38.78 C ATOM 1786 C LEU A 754 25.283 10.512 55.775 1.00 37.61 C ATOM 1787 O LEU A 754 26.301 10.843 55.173 1.00 37.43 O ATOM 1788 CB LEU A 754 23.454 9.394 54.491 1.00 38.76 C ATOM 1789 CG LEU A 754 23.816 10.079 53.167 1.00 38.96 C ATOM 1790 CD1 LEU A 754 24.800 9.220 52.387 1.00 39.08 C ATOM 1791 CD2 LEU A 754 22.556 10.317 52.348 1.00 39.05 C ATOM 1792 N PHE A 755 24.718 11.269 56.711 1.00 36.81 N ATOM 1793 CA PHE A 755 25.286 12.560 57.081 1.00 35.66 C ATOM 1794 C PHE A 755 26.645 12.382 57.745 1.00 35.44 C ATOM 1795 O PHE A 755 27.606 13.069 57.391 1.00 35.00 O ATOM 1796 CB PHE A 755 24.356 13.315 58.030 1.00 34.78 C ATOM 1797 CG PHE A 755 24.930 14.610 58.529 1.00 34.38 C ATOM 1798 CD1 PHE A 755 25.197 15.653 57.647 1.00 34.47 C ATOM 1799 CD2 PHE A 755 25.226 14.780 59.876 1.00 33.95 C ATOM 1800 CE1 PHE A 755 25.755 16.849 58.104 1.00 34.35 C ATOM 1801 CE2 PHE A 755 25.783 15.968 60.345 1.00 34.47 C ATOM 1802 CZ PHE A 755 26.049 17.005 59.459 1.00 34.37 C ATOM 1803 N LEU A 756 26.720 11.469 58.713 1.00 34.29 N ATOM 1804 CA LEU A 756 27.976 11.211 59.412 1.00 33.74 C ATOM 1805 C LEU A 756 29.073 10.850 58.410 1.00 32.83 C ATOM 1806 O LEU A 756 30.223 11.241 58.572 1.00 33.03 O ATOM 1807 CB LEU A 756 27.805 10.076 60.435 1.00 34.02 C ATOM 1808 CG LEU A 756 26.907 10.349 61.651 1.00 34.36 C ATOM 1809 CD1 LEU A 756 26.909 9.135 62.583 1.00 34.63 C ATOM 1810 CD2 LEU A 756 27.414 11.575 62.401 1.00 34.55 C ATOM 1811 N ALA A 757 28.709 10.107 57.370 1.00 31.88 N ATOM 1812 CA ALA A 757 29.665 9.714 56.344 1.00 30.86 C ATOM 1813 C ALA A 757 30.117 10.945 55.555 1.00 30.52 C ATOM 1814 O ALA A 757 31.300 11.103 55.252 1.00 29.30 O ATOM 1815 CB ALA A 757 29.034 8.696 55.409 1.00 30.67 C ATOM 1816 N MET A 758 29.165 11.815 55.228 1.00 29.75 N ATOM 1817 CA MET A 758 29.472 13.030 54.480 1.00 29.58 C ATOM 1818 C MET A 758 30.338 13.972 55.316 1.00 28.32 C ATOM 1819 O MET A 758 31.271 14.588 54.799 1.00 27.59 O ATOM 1820 CB MET A 758 28.178 13.736 54.059 1.00 30.87 C ATOM 1821 CG MET A 758 27.353 12.960 53.035 1.00 31.88 C ATOM 1822 SD MET A 758 28.337 12.469 51.611 1.00 34.89 S ATOM 1823 CE MET A 758 28.144 13.836 50.553 1.00 37.46 C ATOM 1824 N LEU A 759 30.028 14.063 56.609 1.00 26.74 N ATOM 1825 CA LEU A 759 30.765 14.916 57.536 1.00 26.28 C ATOM 1826 C LEU A 759 32.212 14.446 57.665 1.00 25.07 C ATOM 1827 O LEU A 759 33.137 15.256 57.633 1.00 24.51 O ATOM 1828 CB LEU A 759 30.087 14.916 58.914 1.00 25.35 C ATOM 1829 CG LEU A 759 30.770 15.719 60.024 1.00 26.66 C ATOM 1830 CD1 LEU A 759 30.903 17.173 59.608 1.00 27.86 C ATOM 1831 CD2 LEU A 759 29.966 15.601 61.311 1.00 27.35 C ATOM 1832 N MET A 760 32.402 13.137 57.811 1.00 23.76 N ATOM 1833 CA MET A 760 33.745 12.582 57.923 1.00 22.68 C ATOM 1834 C MET A 760 34.556 12.989 56.706 1.00 22.10 C ATOM 1835 O MET A 760 35.694 13.425 56.835 1.00 21.70 O ATOM 1836 CB MET A 760 33.696 11.054 58.019 1.00 22.55 C ATOM 1837 CG MET A 760 33.357 10.521 59.404 1.00 21.17 C ATOM 1838 SD MET A 760 34.662 10.880 60.626 1.00 22.97 S ATOM 1839 CE MET A 760 36.068 9.954 59.967 1.00 21.20 C ATOM 1840 N THR A 761 33.967 12.848 55.522 1.00 21.82 N ATOM 1841 CA THR A 761 34.658 13.212 54.290 1.00 22.25 C ATOM 1842 C THR A 761 35.010 14.698 54.295 1.00 22.43 C ATOM 1843 O THR A 761 36.110 15.090 53.900 1.00 22.77 O ATOM 1844 CB THR A 761 33.791 12.907 53.043 1.00 22.27 C ATOM 1845 OG1 THR A 761 33.520 11.498 52.983 1.00 22.04 O ATOM 1846 CG2 THR A 761 34.519 13.319 51.778 1.00 21.95 C ATOM 1847 N ALA A 762 34.073 15.519 54.748 1.00 21.79 N ATOM 1848 CA ALA A 762 34.296 16.959 54.793 1.00 22.62 C ATOM 1849 C ALA A 762 35.512 17.317 55.649 1.00 22.58 C ATOM 1850 O ALA A 762 36.273 18.217 55.300 1.00 22.27 O ATOM 1851 CB ALA A 762 33.047 17.670 55.319 1.00 20.62 C ATOM 1852 N CYS A 763 35.691 16.615 56.767 1.00 22.42 N ATOM 1853 CA CYS A 763 36.823 16.879 57.649 1.00 22.05 C ATOM 1854 C CYS A 763 38.119 16.319 57.076 1.00 22.14 C ATOM 1855 O CYS A 763 39.185 16.927 57.216 1.00 20.72 O ATOM 1856 CB CYS A 763 36.568 16.278 59.035 1.00 22.36 C ATOM 1857 SG CYS A 763 35.197 17.050 59.958 1.00 23.87 S ATOM 1858 N ASP A 764 38.011 15.173 56.409 1.00 21.35 N ATOM 1859 CA ASP A 764 39.152 14.490 55.810 1.00 21.95 C ATOM 1860 C ASP A 764 39.784 15.317 54.693 1.00 22.80 C ATOM 1861 O ASP A 764 40.992 15.241 54.448 1.00 22.34 O ATOM 1862 CB ASP A 764 38.680 13.157 55.232 1.00 21.79 C ATOM 1863 CG ASP A 764 39.803 12.134 55.049 1.00 23.38 C ATOM 1864 OD1 ASP A 764 40.994 12.439 55.300 1.00 21.60 O ATOM 1865 OD2 ASP A 764 39.459 10.997 54.646 1.00 22.81 O ATOM 1866 N LEU A 765 38.958 16.106 54.016 1.00 23.09 N ATOM 1867 CA LEU A 765 39.423 16.924 52.903 1.00 23.90 C ATOM 1868 C LEU A 765 39.593 18.396 53.274 1.00 23.81 C ATOM 1869 O LEU A 765 39.971 19.210 52.429 1.00 24.79 O ATOM 1870 CB LEU A 765 38.428 16.824 51.735 1.00 24.84 C ATOM 1871 CG LEU A 765 37.966 15.430 51.313 1.00 24.92 C ATOM 1872 CD1 LEU A 765 36.919 15.552 50.218 1.00 26.17 C ATOM 1873 CD2 LEU A 765 39.147 14.616 50.842 1.00 24.09 C ATOM 1874 N SER A 766 39.335 18.735 54.531 1.00 23.43 N ATOM 1875 CA SER A 766 39.394 20.128 54.974 1.00 23.76 C ATOM 1876 C SER A 766 40.687 20.928 54.744 1.00 23.80 C ATOM 1877 O SER A 766 40.668 22.150 54.836 1.00 23.46 O ATOM 1878 CB SER A 766 38.980 20.232 56.446 1.00 23.41 C ATOM 1879 OG SER A 766 39.903 19.578 57.289 1.00 25.01 O ATOM 1880 N ALA A 767 41.800 20.268 54.442 1.00 22.76 N ATOM 1881 CA ALA A 767 43.032 21.020 54.186 1.00 23.49 C ATOM 1882 C ALA A 767 42.794 21.927 52.983 1.00 23.36 C ATOM 1883 O ALA A 767 43.441 22.962 52.831 1.00 23.54 O ATOM 1884 CB ALA A 767 44.192 20.075 53.892 1.00 22.25 C ATOM 1885 N ILE A 768 41.849 21.526 52.134 1.00 23.21 N ATOM 1886 CA ILE A 768 41.523 22.268 50.923 1.00 23.09 C ATOM 1887 C ILE A 768 40.799 23.585 51.208 1.00 23.40 C ATOM 1888 O ILE A 768 40.676 24.437 50.323 1.00 24.42 O ATOM 1889 CB ILE A 768 40.645 21.396 49.967 1.00 23.10 C ATOM 1890 CG1 ILE A 768 40.699 21.951 48.544 1.00 22.94 C ATOM 1891 CG2 ILE A 768 39.189 21.386 50.438 1.00 22.45 C ATOM 1892 CD1 ILE A 768 42.063 21.858 47.892 1.00 22.32 C ATOM 1893 N THR A 769 40.348 23.765 52.447 1.00 23.44 N ATOM 1894 CA THR A 769 39.606 24.964 52.836 1.00 23.84 C ATOM 1895 C THR A 769 40.414 26.014 53.600 1.00 24.61 C ATOM 1896 O THR A 769 39.916 27.109 53.881 1.00 24.77 O ATOM 1897 CB THR A 769 38.429 24.593 53.741 1.00 23.54 C ATOM 1898 OG1 THR A 769 38.927 24.293 55.053 1.00 23.45 O ATOM 1899 CG2 THR A 769 37.696 23.368 53.194 1.00 24.03 C ATOM 1900 N LYS A 770 41.650 25.680 53.947 1.00 24.82 N ATOM 1901 CA LYS A 770 42.495 26.578 54.731 1.00 23.96 C ATOM 1902 C LYS A 770 42.933 27.878 54.053 1.00 24.59 C ATOM 1903 O LYS A 770 42.857 28.020 52.830 1.00 24.15 O ATOM 1904 CB LYS A 770 43.745 25.816 55.188 1.00 24.10 C ATOM 1905 CG LYS A 770 43.456 24.575 56.028 1.00 22.84 C ATOM 1906 CD LYS A 770 42.815 24.936 57.359 1.00 22.59 C ATOM 1907 CE LYS A 770 42.421 23.679 58.134 1.00 22.33 C ATOM 1908 NZ LYS A 770 42.054 23.970 59.543 1.00 19.86 N ATOM 1909 N PRO A 771 43.389 28.856 54.856 1.00 24.70 N ATOM 1910 CA PRO A 771 43.851 30.132 54.306 1.00 25.27 C ATOM 1911 C PRO A 771 44.907 29.845 53.244 1.00 25.72 C ATOM 1912 O PRO A 771 45.697 28.906 53.376 1.00 26.69 O ATOM 1913 CB PRO A 771 44.434 30.835 55.528 1.00 25.87 C ATOM 1914 CG PRO A 771 43.486 30.411 56.613 1.00 25.42 C ATOM 1915 CD PRO A 771 43.289 28.922 56.327 1.00 24.17 C ATOM 1916 N TRP A 772 44.920 30.660 52.198 1.00 26.14 N ATOM 1917 CA TRP A 772 45.853 30.497 51.091 1.00 26.83 C ATOM 1918 C TRP A 772 47.285 30.095 51.458 1.00 27.31 C ATOM 1919 O TRP A 772 47.818 29.132 50.913 1.00 27.44 O ATOM 1920 CB TRP A 772 45.876 31.776 50.254 1.00 27.19 C ATOM 1921 CG TRP A 772 46.790 31.719 49.065 1.00 28.34 C ATOM 1922 CD1 TRP A 772 47.695 32.668 48.686 1.00 28.69 C ATOM 1923 CD2 TRP A 772 46.851 30.685 48.077 1.00 28.48 C ATOM 1924 NE1 TRP A 772 48.316 32.291 47.518 1.00 29.86 N ATOM 1925 CE2 TRP A 772 47.815 31.079 47.120 1.00 28.99 C ATOM 1926 CE3 TRP A 772 46.182 29.465 47.899 1.00 28.49 C ATOM 1927 CZ2 TRP A 772 48.133 30.293 46.006 1.00 28.84 C ATOM 1928 CZ3 TRP A 772 46.497 28.682 46.789 1.00 28.20 C ATOM 1929 CH2 TRP A 772 47.462 29.102 45.857 1.00 28.53 C ATOM 1930 N PRO A 773 47.942 30.833 52.369 1.00 28.12 N ATOM 1931 CA PRO A 773 49.310 30.405 52.681 1.00 27.93 C ATOM 1932 C PRO A 773 49.380 28.998 53.272 1.00 27.54 C ATOM 1933 O PRO A 773 50.331 28.261 53.011 1.00 28.71 O ATOM 1934 CB PRO A 773 49.817 31.499 53.630 1.00 28.56 C ATOM 1935 CG PRO A 773 48.569 32.048 54.246 1.00 29.60 C ATOM 1936 CD PRO A 773 47.585 32.063 53.099 1.00 28.63 C ATOM 1937 N ILE A 774 48.372 28.617 54.050 1.00 26.28 N ATOM 1938 CA ILE A 774 48.354 27.283 54.641 1.00 25.88 C ATOM 1939 C ILE A 774 48.061 26.240 53.560 1.00 25.68 C ATOM 1940 O ILE A 774 48.796 25.266 53.422 1.00 26.17 O ATOM 1941 CB ILE A 774 47.300 27.173 55.765 1.00 25.43 C ATOM 1942 CG1 ILE A 774 47.580 28.225 56.844 1.00 26.53 C ATOM 1943 CG2 ILE A 774 47.328 25.771 56.383 1.00 26.22 C ATOM 1944 CD1 ILE A 774 49.002 28.193 57.366 1.00 26.44 C ATOM 1945 N GLN A 775 47.003 26.451 52.781 1.00 24.76 N ATOM 1946 CA GLN A 775 46.655 25.505 51.718 1.00 24.63 C ATOM 1947 C GLN A 775 47.818 25.287 50.744 1.00 24.83 C ATOM 1948 O GLN A 775 48.084 24.159 50.341 1.00 25.04 O ATOM 1949 CB GLN A 775 45.395 25.978 50.973 1.00 23.00 C ATOM 1950 CG GLN A 775 45.254 25.480 49.528 1.00 23.61 C ATOM 1951 CD GLN A 775 45.246 23.960 49.380 1.00 23.19 C ATOM 1952 OE1 GLN A 775 45.396 23.442 48.270 1.00 24.48 O ATOM 1953 NE2 GLN A 775 45.068 23.245 50.486 1.00 23.08 N ATOM 1954 N GLN A 776 48.519 26.354 50.367 1.00 24.83 N ATOM 1955 CA GLN A 776 49.661 26.198 49.462 1.00 25.79 C ATOM 1956 C GLN A 776 50.624 25.148 50.009 1.00 25.59 C ATOM 1957 O GLN A 776 51.164 24.340 49.256 1.00 26.19 O ATOM 1958 CB GLN A 776 50.429 27.519 49.300 1.00 27.64 C ATOM 1959 CG GLN A 776 49.844 28.494 48.290 1.00 29.97 C ATOM 1960 CD GLN A 776 50.691 29.758 48.154 1.00 32.05 C ATOM 1961 OE1 GLN A 776 50.765 30.570 49.075 1.00 32.82 O ATOM 1962 NE2 GLN A 776 51.341 29.918 47.006 1.00 32.20 N ATOM 1963 N ARG A 777 50.833 25.177 51.323 1.00 25.10 N ATOM 1964 CA ARG A 777 51.730 24.242 51.999 1.00 25.97 C ATOM 1965 C ARG A 777 51.163 22.827 52.052 1.00 25.04 C ATOM 1966 O ARG A 777 51.861 21.857 51.763 1.00 24.81 O ATOM 1967 CB ARG A 777 52.004 24.715 53.430 1.00 26.87 C ATOM 1968 CG ARG A 777 52.721 26.059 53.525 1.00 29.79 C ATOM 1969 CD ARG A 777 54.098 25.974 52.908 1.00 31.69 C ATOM 1970 NE ARG A 777 54.945 27.103 53.292 1.00 33.06 N ATOM 1971 CZ ARG A 777 56.180 27.279 52.836 1.00 34.14 C ATOM 1972 NH1 ARG A 777 56.695 26.399 51.986 1.00 33.83 N ATOM 1973 NH2 ARG A 777 56.899 28.327 53.224 1.00 34.38 N ATOM 1974 N LEU A 778 49.899 22.706 52.438 1.00 25.14 N ATOM 1975 CA LEU A 778 49.287 21.384 52.514 1.00 25.15 C ATOM 1976 C LEU A 778 49.233 20.733 51.130 1.00 25.09 C ATOM 1977 O LEU A 778 49.294 19.513 51.018 1.00 25.32 O ATOM 1978 CB LEU A 778 47.898 21.491 53.138 1.00 25.35 C ATOM 1979 CG LEU A 778 47.949 22.043 54.572 1.00 24.73 C ATOM 1980 CD1 LEU A 778 46.541 22.299 55.083 1.00 23.82 C ATOM 1981 CD2 LEU A 778 48.692 21.061 55.484 1.00 24.45 C ATOM 1982 N ALA A 779 49.128 21.545 50.079 1.00 25.66 N ATOM 1983 CA ALA A 779 49.104 21.015 48.718 1.00 26.21 C ATOM 1984 C ALA A 779 50.495 20.454 48.385 1.00 26.90 C ATOM 1985 O ALA A 779 50.605 19.458 47.675 1.00 27.42 O ATOM 1986 CB ALA A 779 48.706 22.112 47.716 1.00 25.68 C ATOM 1987 N GLU A 780 51.548 21.090 48.904 1.00 27.64 N ATOM 1988 CA GLU A 780 52.926 20.619 48.691 1.00 28.52 C ATOM 1989 C GLU A 780 53.046 19.188 49.220 1.00 28.83 C ATOM 1990 O GLU A 780 53.605 18.310 48.565 1.00 28.91 O ATOM 1991 CB GLU A 780 53.942 21.455 49.481 1.00 30.08 C ATOM 1992 CG GLU A 780 54.454 22.748 48.866 1.00 32.45 C ATOM 1993 CD GLU A 780 55.577 23.365 49.717 1.00 32.89 C ATOM 1994 OE1 GLU A 780 56.720 22.851 49.685 1.00 33.15 O ATOM 1995 OE2 GLU A 780 55.310 24.353 50.433 1.00 33.06 O ATOM 1996 N LEU A 781 52.545 18.980 50.435 1.00 28.04 N ATOM 1997 CA LEU A 781 52.591 17.671 51.072 1.00 28.52 C ATOM 1998 C LEU A 781 51.865 16.628 50.234 1.00 28.15 C ATOM 1999 O LEU A 781 52.397 15.547 49.985 1.00 29.64 O ATOM 2000 CB LEU A 781 51.981 17.740 52.482 1.00 27.20 C ATOM 2001 CG LEU A 781 52.760 18.567 53.513 1.00 27.06 C ATOM 2002 CD1 LEU A 781 52.082 18.489 54.878 1.00 26.98 C ATOM 2003 CD2 LEU A 781 54.187 18.049 53.603 1.00 27.19 C ATOM 2004 N VAL A 782 50.653 16.951 49.798 1.00 28.45 N ATOM 2005 CA VAL A 782 49.882 16.026 48.978 1.00 28.00 C ATOM 2006 C VAL A 782 50.636 15.698 47.697 1.00 28.90 C ATOM 2007 O VAL A 782 50.710 14.535 47.288 1.00 28.29 O ATOM 2008 CB VAL A 782 48.514 16.607 48.616 1.00 28.09 C ATOM 2009 CG1 VAL A 782 47.821 15.703 47.599 1.00 28.37 C ATOM 2010 CG2 VAL A 782 47.665 16.742 49.868 1.00 27.74 C ATOM 2011 N ALA A 783 51.199 16.724 47.065 1.00 29.36 N ATOM 2012 CA ALA A 783 51.955 16.531 45.834 1.00 29.79 C ATOM 2013 C ALA A 783 53.180 15.650 46.079 1.00 29.89 C ATOM 2014 O ALA A 783 53.519 14.816 45.251 1.00 29.22 O ATOM 2015 CB ALA A 783 52.382 17.879 45.261 1.00 30.24 C ATOM 2016 N THR A 784 53.846 15.835 47.214 1.00 30.89 N ATOM 2017 CA THR A 784 55.024 15.028 47.523 1.00 32.31 C ATOM 2018 C THR A 784 54.656 13.552 47.623 1.00 32.78 C ATOM 2019 O THR A 784 55.307 12.702 47.018 1.00 32.20 O ATOM 2020 CB THR A 784 55.683 15.466 48.846 1.00 32.85 C ATOM 2021 OG1 THR A 784 56.255 16.767 48.686 1.00 34.10 O ATOM 2022 CG2 THR A 784 56.778 14.487 49.255 1.00 33.26 C ATOM 2023 N GLU A 785 53.605 13.251 48.377 1.00 33.35 N ATOM 2024 CA GLU A 785 53.175 11.869 48.541 1.00 35.30 C ATOM 2025 C GLU A 785 52.725 11.257 47.216 1.00 36.57 C ATOM 2026 O GLU A 785 52.991 10.088 46.950 1.00 36.40 O ATOM 2027 CB GLU A 785 52.033 11.775 49.561 1.00 34.05 C ATOM 2028 CG GLU A 785 51.800 10.361 50.080 1.00 34.14 C ATOM 2029 CD GLU A 785 50.639 10.265 51.062 1.00 32.63 C ATOM 2030 OE1 GLU A 785 50.602 11.044 52.036 1.00 32.13 O ATOM 2031 OE2 GLU A 785 49.769 9.397 50.860 1.00 33.18 O ATOM 2032 N PHE A 786 52.054 12.055 46.390 1.00 38.52 N ATOM 2033 CA PHE A 786 51.554 11.591 45.096 1.00 41.56 C ATOM 2034 C PHE A 786 52.621 11.341 44.034 1.00 43.37 C ATOM 2035 O PHE A 786 52.622 10.303 43.375 1.00 43.15 O ATOM 2036 CB PHE A 786 50.559 12.598 44.510 1.00 42.76 C ATOM 2037 CG PHE A 786 49.157 12.462 45.029 1.00 44.06 C ATOM 2038 CD1 PHE A 786 48.105 13.080 44.361 1.00 44.13 C ATOM 2039 CD2 PHE A 786 48.881 11.722 46.175 1.00 44.64 C ATOM 2040 CE1 PHE A 786 46.798 12.958 44.819 1.00 45.22 C ATOM 2041 CE2 PHE A 786 47.575 11.594 46.642 1.00 45.06 C ATOM 2042 CZ PHE A 786 46.533 12.213 45.965 1.00 45.01 C ATOM 2043 N PHE A 787 53.522 12.301 43.868 1.00 45.60 N ATOM 2044 CA PHE A 787 54.548 12.211 42.840 1.00 48.34 C ATOM 2045 C PHE A 787 55.933 11.742 43.276 1.00 50.87 C ATOM 2046 O PHE A 787 56.554 10.928 42.596 1.00 51.41 O ATOM 2047 CB PHE A 787 54.664 13.570 42.143 1.00 47.52 C ATOM 2048 CG PHE A 787 53.347 14.119 41.661 1.00 47.05 C ATOM 2049 CD1 PHE A 787 52.628 13.467 40.662 1.00 47.18 C ATOM 2050 CD2 PHE A 787 52.814 15.276 42.220 1.00 46.89 C ATOM 2051 CE1 PHE A 787 51.396 13.961 40.227 1.00 46.97 C ATOM 2052 CE2 PHE A 787 51.584 15.779 41.794 1.00 46.60 C ATOM 2053 CZ PHE A 787 50.874 15.121 40.795 1.00 46.57 C ATOM 2054 N ASP A 788 56.421 12.246 44.403 1.00 54.06 N ATOM 2055 CA ASP A 788 57.754 11.884 44.870 1.00 57.14 C ATOM 2056 C ASP A 788 57.852 10.610 45.702 1.00 59.28 C ATOM 2057 O ASP A 788 58.806 9.842 45.559 1.00 59.70 O ATOM 2058 CB ASP A 788 58.364 13.052 45.648 1.00 57.69 C ATOM 2059 CG ASP A 788 58.480 14.309 44.805 1.00 58.54 C ATOM 2060 OD1 ASP A 788 58.917 14.205 43.640 1.00 59.09 O ATOM 2061 OD2 ASP A 788 58.142 15.400 45.306 1.00 58.96 O ATOM 2062 N GLN A 789 56.879 10.383 46.575 1.00 61.34 N ATOM 2063 CA GLN A 789 56.902 9.190 47.404 1.00 63.39 C ATOM 2064 C GLN A 789 56.356 7.990 46.646 1.00 65.20 C ATOM 2065 O GLN A 789 55.672 8.136 45.633 1.00 65.25 O ATOM 2066 CB GLN A 789 56.098 9.410 48.684 1.00 62.76 C ATOM 2067 CG GLN A 789 56.694 10.450 49.614 1.00 62.27 C ATOM 2068 CD GLN A 789 56.000 10.481 50.959 1.00 61.76 C ATOM 2069 OE1 GLN A 789 54.795 10.708 51.044 1.00 61.40 O ATOM 2070 NE2 GLN A 789 56.759 10.247 52.019 1.00 61.93 N ATOM 2071 N GLY A 790 56.664 6.803 47.150 1.00 67.54 N ATOM 2072 CA GLY A 790 56.218 5.580 46.512 1.00 70.84 C ATOM 2073 C GLY A 790 57.389 4.626 46.449 1.00 73.06 C ATOM 2074 O GLY A 790 57.303 3.547 45.863 1.00 73.36 O ATOM 2075 N ASP A 791 58.489 5.049 47.068 1.00 75.27 N ATOM 2076 CA ASP A 791 59.732 4.286 47.132 1.00 77.61 C ATOM 2077 C ASP A 791 60.847 5.284 47.436 1.00 79.03 C ATOM 2078 O ASP A 791 62.018 5.044 47.136 1.00 79.33 O ATOM 2079 CB ASP A 791 60.009 3.590 45.795 1.00 78.12 C ATOM 2080 CG ASP A 791 60.995 2.445 45.925 1.00 78.59 C ATOM 2081 OD1 ASP A 791 62.162 2.696 46.292 1.00 78.93 O ATOM 2082 OD2 ASP A 791 60.599 1.289 45.662 1.00 78.84 O ATOM 2083 N ARG A 792 60.467 6.410 48.035 1.00 80.51 N ATOM 2084 CA ARG A 792 61.416 7.464 48.373 1.00 81.86 C ATOM 2085 C ARG A 792 61.651 7.520 49.880 1.00 82.34 C ATOM 2086 O ARG A 792 61.064 6.680 50.597 1.00 82.64 O ATOM 2087 CB ARG A 792 60.886 8.815 47.881 1.00 82.55 C ATOM 2088 CG ARG A 792 61.955 9.879 47.687 1.00 83.72 C ATOM 2089 CD ARG A 792 61.346 11.189 47.200 1.00 84.60 C ATOM 2090 NE ARG A 792 62.355 12.121 46.700 1.00 85.24 N ATOM 2091 CZ ARG A 792 63.378 12.581 47.415 1.00 85.49 C ATOM 2092 NH1 ARG A 792 63.541 12.198 48.676 1.00 85.43 N ATOM 2093 NH2 ARG A 792 64.242 13.426 46.868 1.00 85.64 N ATOM 2094 N GLU A 808 58.424 19.870 35.550 1.00 68.21 N ATOM 2095 CA GLU A 808 58.478 20.251 36.990 1.00 68.12 C ATOM 2096 C GLU A 808 57.266 19.712 37.747 1.00 67.56 C ATOM 2097 O GLU A 808 56.231 19.407 37.152 1.00 67.80 O ATOM 2098 CB GLU A 808 58.542 21.777 37.126 1.00 68.77 C ATOM 2099 CG GLU A 808 58.544 22.291 38.561 1.00 69.54 C ATOM 2100 CD GLU A 808 59.771 21.857 39.344 1.00 69.98 C ATOM 2101 OE1 GLU A 808 59.975 20.636 39.517 1.00 70.30 O ATOM 2102 OE2 GLU A 808 60.532 22.743 39.787 1.00 70.46 O ATOM 2103 N LYS A 809 57.408 19.600 39.063 1.00 66.63 N ATOM 2104 CA LYS A 809 56.345 19.096 39.922 1.00 65.43 C ATOM 2105 C LYS A 809 55.273 20.151 40.185 1.00 64.30 C ATOM 2106 O LYS A 809 54.095 19.932 39.903 1.00 64.32 O ATOM 2107 CB LYS A 809 56.944 18.629 41.247 1.00 66.00 C ATOM 2108 CG LYS A 809 55.944 18.060 42.230 1.00 66.61 C ATOM 2109 CD LYS A 809 56.626 17.784 43.553 1.00 67.03 C ATOM 2110 CE LYS A 809 55.665 17.211 44.569 1.00 67.48 C ATOM 2111 NZ LYS A 809 56.328 17.090 45.896 1.00 68.14 N ATOM 2112 N LYS A 810 55.690 21.293 40.725 1.00 62.99 N ATOM 2113 CA LYS A 810 54.778 22.391 41.037 1.00 61.43 C ATOM 2114 C LYS A 810 53.679 22.606 40.004 1.00 59.84 C ATOM 2115 O LYS A 810 52.496 22.601 40.339 1.00 59.74 O ATOM 2116 CB LYS A 810 55.551 23.705 41.197 1.00 62.32 C ATOM 2117 CG LYS A 810 56.206 23.916 42.552 1.00 62.82 C ATOM 2118 CD LYS A 810 56.710 25.349 42.673 1.00 63.50 C ATOM 2119 CE LYS A 810 57.278 25.645 44.053 1.00 63.79 C ATOM 2120 NZ LYS A 810 57.709 27.068 44.170 1.00 63.81 N ATOM 2121 N ASN A 811 54.081 22.801 38.752 1.00 57.79 N ATOM 2122 CA ASN A 811 53.145 23.048 37.658 1.00 55.64 C ATOM 2123 C ASN A 811 51.922 22.137 37.662 1.00 53.52 C ATOM 2124 O ASN A 811 50.825 22.555 37.289 1.00 53.28 O ATOM 2125 CB ASN A 811 53.870 22.918 36.320 1.00 56.32 C ATOM 2126 CG ASN A 811 55.196 23.644 36.311 1.00 57.26 C ATOM 2127 OD1 ASN A 811 55.287 24.807 36.713 1.00 57.51 O ATOM 2128 ND2 ASN A 811 56.237 22.964 35.848 1.00 57.93 N ATOM 2129 N LYS A 812 52.112 20.893 38.084 1.00 51.40 N ATOM 2130 CA LYS A 812 51.017 19.932 38.126 1.00 48.95 C ATOM 2131 C LYS A 812 50.053 20.236 39.269 1.00 46.54 C ATOM 2132 O LYS A 812 48.877 19.888 39.213 1.00 46.09 O ATOM 2133 CB LYS A 812 51.566 18.514 38.308 1.00 49.74 C ATOM 2134 CG LYS A 812 52.766 18.189 37.440 1.00 50.45 C ATOM 2135 CD LYS A 812 53.125 16.714 37.518 1.00 51.26 C ATOM 2136 CE LYS A 812 52.016 15.848 36.939 1.00 51.82 C ATOM 2137 NZ LYS A 812 52.389 14.407 36.923 1.00 52.77 N ATOM 2138 N ILE A 813 50.555 20.905 40.299 1.00 43.60 N ATOM 2139 CA ILE A 813 49.745 21.211 41.470 1.00 40.82 C ATOM 2140 C ILE A 813 48.517 22.104 41.281 1.00 39.20 C ATOM 2141 O ILE A 813 47.415 21.730 41.675 1.00 37.95 O ATOM 2142 CB ILE A 813 50.633 21.793 42.594 1.00 40.12 C ATOM 2143 CG1 ILE A 813 51.661 20.736 43.015 1.00 39.09 C ATOM 2144 CG2 ILE A 813 49.773 22.236 43.771 1.00 38.50 C ATOM 2145 CD1 ILE A 813 52.609 21.180 44.101 1.00 40.56 C ATOM 2146 N PRO A 814 48.680 23.287 40.669 1.00 38.45 N ATOM 2147 CA PRO A 814 47.507 24.150 40.493 1.00 37.62 C ATOM 2148 C PRO A 814 46.319 23.508 39.777 1.00 36.63 C ATOM 2149 O PRO A 814 45.187 23.603 40.250 1.00 36.40 O ATOM 2150 CB PRO A 814 48.076 25.373 39.761 1.00 38.21 C ATOM 2151 CG PRO A 814 49.284 24.834 39.061 1.00 38.72 C ATOM 2152 CD PRO A 814 49.881 23.890 40.067 1.00 38.23 C ATOM 2153 N SER A 815 46.563 22.846 38.653 1.00 36.02 N ATOM 2154 CA SER A 815 45.466 22.207 37.935 1.00 36.04 C ATOM 2155 C SER A 815 44.908 21.029 38.748 1.00 34.95 C ATOM 2156 O SER A 815 43.703 20.782 38.744 1.00 34.97 O ATOM 2157 CB SER A 815 45.926 21.739 36.549 1.00 36.34 C ATOM 2158 OG SER A 815 46.955 20.773 36.643 1.00 38.30 O ATOM 2159 N MET A 816 45.782 20.318 39.456 1.00 33.91 N ATOM 2160 CA MET A 816 45.354 19.189 40.279 1.00 32.25 C ATOM 2161 C MET A 816 44.401 19.658 41.367 1.00 31.26 C ATOM 2162 O MET A 816 43.357 19.041 41.596 1.00 30.42 O ATOM 2163 CB MET A 816 46.550 18.494 40.935 1.00 32.87 C ATOM 2164 CG MET A 816 46.141 17.454 41.986 1.00 34.57 C ATOM 2165 SD MET A 816 47.517 16.558 42.741 1.00 36.00 S ATOM 2166 CE MET A 816 48.033 17.707 43.994 1.00 34.52 C ATOM 2167 N GLN A 817 44.758 20.754 42.035 1.00 29.69 N ATOM 2168 CA GLN A 817 43.922 21.289 43.101 1.00 28.59 C ATOM 2169 C GLN A 817 42.593 21.847 42.593 1.00 28.57 C ATOM 2170 O GLN A 817 41.581 21.779 43.290 1.00 28.10 O ATOM 2171 CB GLN A 817 44.674 22.374 43.881 1.00 28.02 C ATOM 2172 CG GLN A 817 45.871 21.865 44.677 1.00 27.10 C ATOM 2173 CD GLN A 817 45.533 20.672 45.557 1.00 27.36 C ATOM 2174 OE1 GLN A 817 45.470 19.532 45.085 1.00 26.66 O ATOM 2175 NE2 GLN A 817 45.304 20.929 46.843 1.00 26.57 N ATOM 2176 N VAL A 818 42.589 22.413 41.388 1.00 28.47 N ATOM 2177 CA VAL A 818 41.343 22.945 40.839 1.00 28.28 C ATOM 2178 C VAL A 818 40.435 21.775 40.468 1.00 27.80 C ATOM 2179 O VAL A 818 39.237 21.809 40.717 1.00 28.18 O ATOM 2180 CB VAL A 818 41.586 23.815 39.576 1.00 28.11 C ATOM 2181 CG1 VAL A 818 40.251 24.302 39.020 1.00 28.07 C ATOM 2182 CG2 VAL A 818 42.476 25.021 39.925 1.00 26.93 C ATOM 2183 N GLY A 819 41.018 20.737 39.877 1.00 28.19 N ATOM 2184 CA GLY A 819 40.239 19.574 39.494 1.00 28.11 C ATOM 2185 C GLY A 819 39.664 18.902 40.727 1.00 28.42 C ATOM 2186 O GLY A 819 38.498 18.497 40.742 1.00 26.66 O ATOM 2187 N PHE A 820 40.490 18.799 41.769 1.00 28.31 N ATOM 2188 CA PHE A 820 40.075 18.188 43.028 1.00 28.49 C ATOM 2189 C PHE A 820 38.898 18.947 43.615 1.00 28.60 C ATOM 2190 O PHE A 820 37.899 18.349 44.019 1.00 28.01 O ATOM 2191 CB PHE A 820 41.238 18.189 44.028 1.00 29.06 C ATOM 2192 CG PHE A 820 40.879 17.643 45.386 1.00 29.18 C ATOM 2193 CD1 PHE A 820 40.457 16.325 45.532 1.00 29.38 C ATOM 2194 CD2 PHE A 820 40.982 18.442 46.521 1.00 29.72 C ATOM 2195 CE1 PHE A 820 40.141 15.803 46.793 1.00 29.10 C ATOM 2196 CE2 PHE A 820 40.671 17.932 47.788 1.00 30.75 C ATOM 2197 CZ PHE A 820 40.248 16.605 47.918 1.00 29.62 C ATOM 2198 N ILE A 821 39.011 20.273 43.652 1.00 29.21 N ATOM 2199 CA ILE A 821 37.948 21.107 44.195 1.00 29.50 C ATOM 2200 C ILE A 821 36.635 21.011 43.414 1.00 30.03 C ATOM 2201 O ILE A 821 35.573 20.837 44.000 1.00 30.31 O ATOM 2202 CB ILE A 821 38.384 22.592 44.256 1.00 29.95 C ATOM 2203 CG1 ILE A 821 39.488 22.765 45.305 1.00 30.09 C ATOM 2204 CG2 ILE A 821 37.190 23.478 44.612 1.00 29.13 C ATOM 2205 CD1 ILE A 821 40.056 24.164 45.371 1.00 30.52 C ATOM 2206 N ASP A 822 36.702 21.126 42.094 1.00 31.42 N ATOM 2207 CA ASP A 822 35.487 21.057 41.282 1.00 32.40 C ATOM 2208 C ASP A 822 34.789 19.713 41.406 1.00 32.69 C ATOM 2209 O ASP A 822 33.620 19.624 41.782 1.00 33.09 O ATOM 2210 CB ASP A 822 35.805 21.299 39.804 1.00 33.16 C ATOM 2211 CG ASP A 822 36.085 22.753 39.497 1.00 33.88 C ATOM 2212 OD1 ASP A 822 35.416 23.619 40.097 1.00 34.43 O ATOM 2213 OD2 ASP A 822 36.958 23.026 38.647 1.00 35.03 O ATOM 2214 N ALA A 823 35.538 18.666 41.099 1.00 32.10 N ATOM 2215 CA ALA A 823 35.020 17.315 41.113 1.00 32.83 C ATOM 2216 C ALA A 823 34.624 16.703 42.457 1.00 32.44 C ATOM 2217 O ALA A 823 33.644 15.963 42.521 1.00 32.63 O ATOM 2218 CB ALA A 823 36.008 16.422 40.413 1.00 32.28 C ATOM 2219 N ILE A 824 35.351 17.020 43.527 1.00 32.23 N ATOM 2220 CA ILE A 824 35.068 16.430 44.841 1.00 32.19 C ATOM 2221 C ILE A 824 34.624 17.331 45.996 1.00 32.32 C ATOM 2222 O ILE A 824 33.722 16.966 46.760 1.00 31.86 O ATOM 2223 CB ILE A 824 36.305 15.630 45.352 1.00 32.99 C ATOM 2224 CG1 ILE A 824 36.510 14.375 44.507 1.00 34.20 C ATOM 2225 CG2 ILE A 824 36.114 15.224 46.819 1.00 33.64 C ATOM 2226 CD1 ILE A 824 35.409 13.338 44.676 1.00 34.79 C ATOM 2227 N CYS A 825 35.248 18.498 46.127 1.00 31.92 N ATOM 2228 CA CYS A 825 34.973 19.397 47.248 1.00 31.49 C ATOM 2229 C CYS A 825 33.821 20.403 47.228 1.00 32.05 C ATOM 2230 O CYS A 825 33.062 20.493 48.196 1.00 31.20 O ATOM 2231 CB CYS A 825 36.263 20.148 47.585 1.00 31.20 C ATOM 2232 SG CYS A 825 37.694 19.058 47.751 1.00 30.53 S ATOM 2233 N LEU A 826 33.700 21.179 46.157 1.00 32.58 N ATOM 2234 CA LEU A 826 32.656 22.198 46.103 1.00 33.94 C ATOM 2235 C LEU A 826 31.258 21.751 46.534 1.00 34.41 C ATOM 2236 O LEU A 826 30.671 22.337 47.447 1.00 34.06 O ATOM 2237 CB LEU A 826 32.612 22.826 44.708 1.00 34.32 C ATOM 2238 CG LEU A 826 33.841 23.707 44.432 1.00 35.28 C ATOM 2239 CD1 LEU A 826 33.755 24.311 43.032 1.00 34.77 C ATOM 2240 CD2 LEU A 826 33.920 24.816 45.488 1.00 35.22 C ATOM 2241 N GLN A 827 30.726 20.715 45.897 1.00 34.80 N ATOM 2242 CA GLN A 827 29.393 20.241 46.247 1.00 35.63 C ATOM 2243 C GLN A 827 29.253 19.800 47.702 1.00 34.41 C ATOM 2244 O GLN A 827 28.238 20.079 48.343 1.00 34.18 O ATOM 2245 CB GLN A 827 28.978 19.094 45.328 1.00 37.53 C ATOM 2246 CG GLN A 827 28.538 19.540 43.949 1.00 41.26 C ATOM 2247 CD GLN A 827 27.842 18.433 43.193 1.00 43.28 C ATOM 2248 OE1 GLN A 827 28.469 17.449 42.795 1.00 45.48 O ATOM 2249 NE2 GLN A 827 26.531 18.575 43.007 1.00 44.13 N ATOM 2250 N LEU A 828 30.267 19.116 48.223 1.00 32.81 N ATOM 2251 CA LEU A 828 30.221 18.644 49.603 1.00 31.56 C ATOM 2252 C LEU A 828 30.097 19.785 50.604 1.00 30.95 C ATOM 2253 O LEU A 828 29.214 19.769 51.462 1.00 30.78 O ATOM 2254 CB LEU A 828 31.463 17.807 49.929 1.00 30.85 C ATOM 2255 CG LEU A 828 31.561 17.294 51.372 1.00 31.10 C ATOM 2256 CD1 LEU A 828 30.290 16.548 51.761 1.00 28.71 C ATOM 2257 CD2 LEU A 828 32.780 16.393 51.502 1.00 29.84 C ATOM 2258 N TYR A 829 30.976 20.780 50.502 1.00 30.25 N ATOM 2259 CA TYR A 829 30.915 21.903 51.428 1.00 29.63 C ATOM 2260 C TYR A 829 29.656 22.746 51.229 1.00 31.03 C ATOM 2261 O TYR A 829 29.161 23.365 52.173 1.00 30.36 O ATOM 2262 CB TYR A 829 32.184 22.750 51.313 1.00 27.51 C ATOM 2263 CG TYR A 829 33.395 22.000 51.817 1.00 25.67 C ATOM 2264 CD1 TYR A 829 34.406 21.599 50.949 1.00 25.70 C ATOM 2265 CD2 TYR A 829 33.500 21.638 53.162 1.00 25.11 C ATOM 2266 CE1 TYR A 829 35.497 20.847 51.410 1.00 25.99 C ATOM 2267 CE2 TYR A 829 34.586 20.887 53.630 1.00 24.96 C ATOM 2268 CZ TYR A 829 35.575 20.499 52.751 1.00 24.77 C ATOM 2269 OH TYR A 829 36.652 19.779 53.211 1.00 26.34 O ATOM 2270 N GLU A 830 29.135 22.764 50.005 1.00 33.02 N ATOM 2271 CA GLU A 830 27.908 23.501 49.739 1.00 35.24 C ATOM 2272 C GLU A 830 26.801 22.760 50.473 1.00 35.34 C ATOM 2273 O GLU A 830 26.015 23.358 51.202 1.00 35.24 O ATOM 2274 CB GLU A 830 27.603 23.544 48.238 1.00 36.80 C ATOM 2275 CG GLU A 830 28.574 24.397 47.437 1.00 40.01 C ATOM 2276 CD GLU A 830 28.138 24.594 45.997 1.00 42.51 C ATOM 2277 OE1 GLU A 830 28.949 25.113 45.200 1.00 44.05 O ATOM 2278 OE2 GLU A 830 26.986 24.237 45.660 1.00 44.51 O ATOM 2279 N ALA A 831 26.763 21.445 50.291 1.00 36.58 N ATOM 2280 CA ALA A 831 25.757 20.612 50.944 1.00 37.09 C ATOM 2281 C ALA A 831 25.815 20.773 52.463 1.00 37.54 C ATOM 2282 O ALA A 831 24.784 20.939 53.121 1.00 38.03 O ATOM 2283 CB ALA A 831 25.967 19.152 50.563 1.00 36.98 C ATOM 2284 N LEU A 832 27.024 20.728 53.015 1.00 37.39 N ATOM 2285 CA LEU A 832 27.213 20.866 54.454 1.00 37.92 C ATOM 2286 C LEU A 832 26.704 22.209 54.973 1.00 38.41 C ATOM 2287 O LEU A 832 26.106 22.285 56.048 1.00 37.66 O ATOM 2288 CB LEU A 832 28.695 20.718 54.798 1.00 37.33 C ATOM 2289 CG LEU A 832 29.120 20.913 56.256 1.00 37.07 C ATOM 2290 CD1 LEU A 832 28.400 19.920 57.163 1.00 36.76 C ATOM 2291 CD2 LEU A 832 30.629 20.727 56.354 1.00 36.13 C ATOM 2292 N THR A 833 26.952 23.267 54.210 1.00 39.40 N ATOM 2293 CA THR A 833 26.521 24.604 54.600 1.00 40.43 C ATOM 2294 C THR A 833 24.999 24.687 54.715 1.00 41.58 C ATOM 2295 O THR A 833 24.475 25.383 55.585 1.00 41.44 O ATOM 2296 CB THR A 833 27.016 25.655 53.591 1.00 40.43 C ATOM 2297 OG1 THR A 833 28.445 25.594 53.508 1.00 40.08 O ATOM 2298 CG2 THR A 833 26.605 27.054 54.029 1.00 40.30 C ATOM 2299 N HIS A 834 24.294 23.977 53.839 1.00 42.83 N ATOM 2300 CA HIS A 834 22.836 23.971 53.871 1.00 44.18 C ATOM 2301 C HIS A 834 22.361 23.317 55.164 1.00 44.18 C ATOM 2302 O HIS A 834 21.443 23.810 55.816 1.00 44.11 O ATOM 2303 CB HIS A 834 22.272 23.210 52.667 1.00 45.90 C ATOM 2304 CG HIS A 834 22.606 23.835 51.348 1.00 47.87 C ATOM 2305 ND1 HIS A 834 22.382 25.169 51.078 1.00 48.91 N ATOM 2306 CD2 HIS A 834 23.132 23.306 50.217 1.00 49.09 C ATOM 2307 CE1 HIS A 834 22.756 25.434 49.839 1.00 49.44 C ATOM 2308 NE2 HIS A 834 23.215 24.322 49.294 1.00 49.70 N ATOM 2309 N VAL A 835 22.995 22.208 55.534 1.00 44.13 N ATOM 2310 CA VAL A 835 22.634 21.509 56.761 1.00 44.29 C ATOM 2311 C VAL A 835 22.879 22.428 57.953 1.00 44.77 C ATOM 2312 O VAL A 835 22.083 22.469 58.891 1.00 44.61 O ATOM 2313 CB VAL A 835 23.459 20.212 56.941 1.00 43.91 C ATOM 2314 CG1 VAL A 835 23.153 19.586 58.287 1.00 43.92 C ATOM 2315 CG2 VAL A 835 23.130 19.227 55.832 1.00 43.83 C ATOM 2316 N SER A 836 23.986 23.164 57.909 1.00 45.46 N ATOM 2317 CA SER A 836 24.328 24.091 58.979 1.00 45.41 C ATOM 2318 C SER A 836 24.998 25.344 58.426 1.00 45.70 C ATOM 2319 O SER A 836 26.135 25.301 57.949 1.00 45.42 O ATOM 2320 CB SER A 836 25.254 23.426 59.992 1.00 45.69 C ATOM 2321 OG SER A 836 25.536 24.312 61.061 1.00 46.80 O ATOM 2322 N GLU A 837 24.278 26.459 58.501 1.00 45.48 N ATOM 2323 CA GLU A 837 24.761 27.745 58.012 1.00 45.20 C ATOM 2324 C GLU A 837 26.150 28.072 58.544 1.00 43.35 C ATOM 2325 O GLU A 837 26.976 28.643 57.840 1.00 43.07 O ATOM 2326 CB GLU A 837 23.805 28.863 58.445 1.00 47.25 C ATOM 2327 CG GLU A 837 22.344 28.658 58.069 1.00 50.33 C ATOM 2328 CD GLU A 837 22.094 28.781 56.579 1.00 51.93 C ATOM 2329 OE1 GLU A 837 22.450 27.842 55.830 1.00 52.30 O ATOM 2330 OE2 GLU A 837 21.546 29.826 56.159 1.00 52.90 O ATOM 2331 N ASP A 838 26.394 27.697 59.792 1.00 42.24 N ATOM 2332 CA ASP A 838 27.655 27.979 60.462 1.00 41.10 C ATOM 2333 C ASP A 838 28.909 27.278 59.930 1.00 39.59 C ATOM 2334 O ASP A 838 30.019 27.568 60.380 1.00 39.40 O ATOM 2335 CB ASP A 838 27.488 27.714 61.959 1.00 42.20 C ATOM 2336 CG ASP A 838 26.456 28.636 62.599 1.00 43.60 C ATOM 2337 OD1 ASP A 838 26.705 29.861 62.660 1.00 43.79 O ATOM 2338 OD2 ASP A 838 25.394 28.139 63.035 1.00 43.97 O ATOM 2339 N CYS A 839 28.740 26.366 58.978 1.00 37.72 N ATOM 2340 CA CYS A 839 29.877 25.665 58.385 1.00 35.85 C ATOM 2341 C CYS A 839 30.321 26.349 57.099 1.00 35.52 C ATOM 2342 O CYS A 839 31.191 25.852 56.382 1.00 34.97 O ATOM 2343 CB CYS A 839 29.519 24.209 58.094 1.00 35.29 C ATOM 2344 SG CYS A 839 29.469 23.164 59.566 1.00 32.68 S ATOM 2345 N PHE A 840 29.727 27.506 56.826 1.00 35.34 N ATOM 2346 CA PHE A 840 30.033 28.284 55.631 1.00 34.86 C ATOM 2347 C PHE A 840 31.526 28.541 55.402 1.00 33.81 C ATOM 2348 O PHE A 840 32.014 28.410 54.280 1.00 34.50 O ATOM 2349 CB PHE A 840 29.292 29.626 55.681 1.00 36.35 C ATOM 2350 CG PHE A 840 29.591 30.530 54.518 1.00 37.35 C ATOM 2351 CD1 PHE A 840 29.199 30.180 53.230 1.00 37.68 C ATOM 2352 CD2 PHE A 840 30.276 31.727 54.710 1.00 38.31 C ATOM 2353 CE1 PHE A 840 29.484 31.011 52.140 1.00 38.62 C ATOM 2354 CE2 PHE A 840 30.568 32.566 53.628 1.00 38.81 C ATOM 2355 CZ PHE A 840 30.170 32.205 52.340 1.00 38.43 C ATOM 2356 N PRO A 841 32.268 28.922 56.457 1.00 32.73 N ATOM 2357 CA PRO A 841 33.698 29.181 56.262 1.00 32.14 C ATOM 2358 C PRO A 841 34.476 28.072 55.562 1.00 31.60 C ATOM 2359 O PRO A 841 35.456 28.349 54.867 1.00 31.70 O ATOM 2360 CB PRO A 841 34.210 29.491 57.677 1.00 32.17 C ATOM 2361 CG PRO A 841 33.108 29.027 58.597 1.00 33.53 C ATOM 2362 CD PRO A 841 31.853 29.270 57.824 1.00 33.24 C ATOM 2363 N LEU A 842 34.048 26.822 55.731 1.00 30.04 N ATOM 2364 CA LEU A 842 34.722 25.718 55.059 1.00 28.62 C ATOM 2365 C LEU A 842 34.519 25.912 53.559 1.00 28.71 C ATOM 2366 O LEU A 842 35.456 25.805 52.771 1.00 28.43 O ATOM 2367 CB LEU A 842 34.136 24.371 55.504 1.00 27.39 C ATOM 2368 CG LEU A 842 34.498 23.915 56.924 1.00 27.17 C ATOM 2369 CD1 LEU A 842 33.683 22.676 57.309 1.00 26.91 C ATOM 2370 CD2 LEU A 842 35.985 23.618 56.996 1.00 26.62 C ATOM 2371 N LEU A 843 33.281 26.210 53.176 1.00 29.02 N ATOM 2372 CA LEU A 843 32.936 26.433 51.778 1.00 28.68 C ATOM 2373 C LEU A 843 33.623 27.688 51.250 1.00 28.97 C ATOM 2374 O LEU A 843 34.201 27.682 50.170 1.00 29.07 O ATOM 2375 CB LEU A 843 31.422 26.579 51.637 1.00 28.84 C ATOM 2376 CG LEU A 843 30.910 27.102 50.295 1.00 28.49 C ATOM 2377 CD1 LEU A 843 31.326 26.158 49.181 1.00 29.41 C ATOM 2378 CD2 LEU A 843 29.389 27.236 50.356 1.00 29.84 C ATOM 2379 N ASP A 844 33.556 28.767 52.021 1.00 30.01 N ATOM 2380 CA ASP A 844 34.191 30.020 51.618 1.00 30.34 C ATOM 2381 C ASP A 844 35.678 29.760 51.370 1.00 29.94 C ATOM 2382 O ASP A 844 36.226 30.115 50.324 1.00 29.66 O ATOM 2383 CB ASP A 844 34.038 31.064 52.722 1.00 31.63 C ATOM 2384 CG ASP A 844 34.342 32.468 52.242 1.00 32.55 C ATOM 2385 OD1 ASP A 844 34.808 33.282 53.060 1.00 34.10 O ATOM 2386 OD2 ASP A 844 34.103 32.759 51.051 1.00 34.53 O ATOM 2387 N GLY A 845 36.322 29.121 52.340 1.00 29.14 N ATOM 2388 CA GLY A 845 37.734 28.830 52.211 1.00 28.15 C ATOM 2389 C GLY A 845 38.049 28.013 50.978 1.00 28.60 C ATOM 2390 O GLY A 845 39.054 28.252 50.310 1.00 28.66 O ATOM 2391 N CYS A 846 37.191 27.045 50.671 1.00 29.05 N ATOM 2392 CA CYS A 846 37.399 26.180 49.517 1.00 29.85 C ATOM 2393 C CYS A 846 37.319 26.979 48.225 1.00 30.11 C ATOM 2394 O CYS A 846 38.080 26.747 47.286 1.00 29.65 O ATOM 2395 CB CYS A 846 36.349 25.068 49.501 1.00 29.98 C ATOM 2396 SG CYS A 846 36.585 23.867 48.186 1.00 30.84 S ATOM 2397 N ARG A 847 36.379 27.913 48.184 1.00 30.81 N ATOM 2398 CA ARG A 847 36.193 28.760 47.012 1.00 32.54 C ATOM 2399 C ARG A 847 37.374 29.701 46.814 1.00 31.90 C ATOM 2400 O ARG A 847 37.880 29.843 45.706 1.00 33.42 O ATOM 2401 CB ARG A 847 34.912 29.569 47.157 1.00 32.77 C ATOM 2402 CG ARG A 847 33.653 28.752 46.957 1.00 34.36 C ATOM 2403 CD ARG A 847 32.442 29.582 47.332 1.00 35.13 C ATOM 2404 NE ARG A 847 31.186 28.936 46.970 1.00 36.07 N ATOM 2405 CZ ARG A 847 30.002 29.342 47.412 1.00 36.29 C ATOM 2406 NH1 ARG A 847 29.933 30.385 48.229 1.00 35.79 N ATOM 2407 NH2 ARG A 847 28.893 28.719 47.032 1.00 35.82 N ATOM 2408 N LYS A 848 37.812 30.344 47.888 1.00 32.70 N ATOM 2409 CA LYS A 848 38.942 31.258 47.791 1.00 32.02 C ATOM 2410 C LYS A 848 40.143 30.514 47.222 1.00 31.83 C ATOM 2411 O LYS A 848 40.835 31.024 46.341 1.00 31.51 O ATOM 2412 CB LYS A 848 39.266 31.849 49.164 1.00 33.25 C ATOM 2413 CG LYS A 848 38.220 32.852 49.643 1.00 34.64 C ATOM 2414 CD LYS A 848 38.535 33.386 51.026 1.00 36.03 C ATOM 2415 CE LYS A 848 37.464 34.366 51.479 1.00 36.82 C ATOM 2416 NZ LYS A 848 37.723 34.905 52.846 1.00 37.75 N ATOM 2417 N ASN A 849 40.374 29.294 47.705 1.00 30.48 N ATOM 2418 CA ASN A 849 41.489 28.501 47.206 1.00 29.64 C ATOM 2419 C ASN A 849 41.335 28.116 45.732 1.00 28.95 C ATOM 2420 O ASN A 849 42.325 28.051 45.000 1.00 27.78 O ATOM 2421 CB ASN A 849 41.685 27.242 48.061 1.00 29.24 C ATOM 2422 CG ASN A 849 42.384 27.540 49.385 1.00 29.87 C ATOM 2423 OD1 ASN A 849 43.260 28.403 49.449 1.00 27.60 O ATOM 2424 ND2 ASN A 849 42.012 26.814 50.438 1.00 28.16 N ATOM 2425 N ARG A 850 40.110 27.854 45.284 1.00 29.49 N ATOM 2426 CA ARG A 850 39.922 27.496 43.875 1.00 30.82 C ATOM 2427 C ARG A 850 40.374 28.679 43.026 1.00 31.76 C ATOM 2428 O ARG A 850 41.099 28.525 42.046 1.00 31.56 O ATOM 2429 CB ARG A 850 38.455 27.190 43.552 1.00 31.11 C ATOM 2430 CG ARG A 850 38.278 26.728 42.112 1.00 32.12 C ATOM 2431 CD ARG A 850 36.822 26.576 41.689 1.00 33.80 C ATOM 2432 NE ARG A 850 36.725 25.949 40.372 1.00 33.21 N ATOM 2433 CZ ARG A 850 37.117 26.510 39.229 1.00 33.14 C ATOM 2434 NH1 ARG A 850 37.638 27.731 39.218 1.00 33.01 N ATOM 2435 NH2 ARG A 850 36.992 25.844 38.090 1.00 32.02 N ATOM 2436 N GLN A 851 39.929 29.862 43.429 1.00 34.17 N ATOM 2437 CA GLN A 851 40.259 31.109 42.754 1.00 36.68 C ATOM 2438 C GLN A 851 41.778 31.283 42.669 1.00 36.44 C ATOM 2439 O GLN A 851 42.323 31.534 41.595 1.00 36.01 O ATOM 2440 CB GLN A 851 39.609 32.260 43.527 1.00 38.87 C ATOM 2441 CG GLN A 851 39.871 33.652 43.001 1.00 43.28 C ATOM 2442 CD GLN A 851 39.024 34.687 43.723 1.00 46.06 C ATOM 2443 OE1 GLN A 851 39.063 34.794 44.954 1.00 48.08 O ATOM 2444 NE2 GLN A 851 38.245 35.449 42.961 1.00 47.71 N ATOM 2445 N LYS A 852 42.459 31.123 43.802 1.00 36.45 N ATOM 2446 CA LYS A 852 43.913 31.255 43.853 1.00 36.02 C ATOM 2447 C LYS A 852 44.643 30.257 42.954 1.00 35.56 C ATOM 2448 O LYS A 852 45.530 30.633 42.191 1.00 34.56 O ATOM 2449 CB LYS A 852 44.407 31.088 45.293 1.00 37.15 C ATOM 2450 CG LYS A 852 44.115 32.270 46.211 1.00 37.95 C ATOM 2451 CD LYS A 852 44.966 33.474 45.836 1.00 39.41 C ATOM 2452 CE LYS A 852 44.877 34.569 46.887 1.00 39.50 C ATOM 2453 NZ LYS A 852 45.703 35.750 46.511 1.00 39.99 N ATOM 2454 N TRP A 853 44.280 28.981 43.050 1.00 35.24 N ATOM 2455 CA TRP A 853 44.927 27.954 42.239 1.00 34.88 C ATOM 2456 C TRP A 853 44.650 28.134 40.746 1.00 36.31 C ATOM 2457 O TRP A 853 45.529 27.908 39.911 1.00 35.72 O ATOM 2458 CB TRP A 853 44.470 26.557 42.676 1.00 33.38 C ATOM 2459 CG TRP A 853 45.115 26.052 43.947 1.00 31.36 C ATOM 2460 CD1 TRP A 853 44.476 25.616 45.077 1.00 30.70 C ATOM 2461 CD2 TRP A 853 46.518 25.890 44.196 1.00 30.28 C ATOM 2462 NE1 TRP A 853 45.394 25.190 46.010 1.00 29.39 N ATOM 2463 CE2 TRP A 853 46.653 25.347 45.497 1.00 30.18 C ATOM 2464 CE3 TRP A 853 47.673 26.149 43.448 1.00 30.35 C ATOM 2465 CZ2 TRP A 853 47.901 25.059 46.065 1.00 29.91 C ATOM 2466 CZ3 TRP A 853 48.918 25.862 44.015 1.00 30.78 C ATOM 2467 CH2 TRP A 853 49.017 25.323 45.313 1.00 30.50 C ATOM 2468 N GLN A 854 43.425 28.534 40.416 1.00 37.83 N ATOM 2469 CA GLN A 854 43.036 28.735 39.023 1.00 39.72 C ATOM 2470 C GLN A 854 43.851 29.874 38.406 1.00 40.20 C ATOM 2471 O GLN A 854 44.315 29.776 37.271 1.00 39.40 O ATOM 2472 CB GLN A 854 41.536 29.038 38.943 1.00 41.25 C ATOM 2473 CG GLN A 854 40.818 28.324 37.800 1.00 44.10 C ATOM 2474 CD GLN A 854 40.919 29.068 36.490 1.00 45.46 C ATOM 2475 OE1 GLN A 854 40.357 30.154 36.335 1.00 47.15 O ATOM 2476 NE2 GLN A 854 41.639 28.491 35.538 1.00 46.05 N ATOM 2477 N ALA A 855 44.026 30.950 39.166 1.00 41.09 N ATOM 2478 CA ALA A 855 44.802 32.086 38.696 1.00 42.40 C ATOM 2479 C ALA A 855 46.216 31.607 38.372 1.00 43.59 C ATOM 2480 O ALA A 855 46.811 32.033 37.386 1.00 43.48 O ATOM 2481 CB ALA A 855 44.841 33.169 39.762 1.00 42.50 C ATOM 2482 N LEU A 856 46.749 30.714 39.202 1.00 44.56 N ATOM 2483 CA LEU A 856 48.088 30.178 38.974 1.00 45.85 C ATOM 2484 C LEU A 856 48.080 29.248 37.767 1.00 47.27 C ATOM 2485 O LEU A 856 49.035 29.217 36.990 1.00 47.18 O ATOM 2486 CB LEU A 856 48.579 29.406 40.201 1.00 45.30 C ATOM 2487 CG LEU A 856 48.934 30.208 41.454 1.00 45.32 C ATOM 2488 CD1 LEU A 856 49.354 29.251 42.559 1.00 45.02 C ATOM 2489 CD2 LEU A 856 50.056 31.190 41.145 1.00 45.53 C ATOM 2490 N ALA A 857 46.999 28.486 37.622 1.00 49.11 N ATOM 2491 CA ALA A 857 46.854 27.552 36.513 1.00 51.82 C ATOM 2492 C ALA A 857 46.979 28.287 35.180 1.00 53.77 C ATOM 2493 O ALA A 857 47.632 27.808 34.253 1.00 53.77 O ATOM 2494 CB ALA A 857 45.505 26.848 36.597 1.00 51.72 C ATOM 2495 N GLU A 858 46.347 29.454 35.095 1.00 55.97 N ATOM 2496 CA GLU A 858 46.393 30.264 33.882 1.00 58.82 C ATOM 2497 C GLU A 858 47.754 30.949 33.811 1.00 60.62 C ATOM 2498 O GLU A 858 48.073 31.623 32.831 1.00 60.99 O ATOM 2499 CB GLU A 858 45.285 31.317 33.913 1.00 58.29 C ATOM 2500 CG GLU A 858 43.945 30.772 34.377 1.00 58.53 C ATOM 2501 CD GLU A 858 42.893 31.848 34.540 1.00 58.51 C ATOM 2502 OE1 GLU A 858 43.238 32.960 34.998 1.00 58.59 O ATOM 2503 OE2 GLU A 858 41.716 31.576 34.226 1.00 58.36 O ATOM 2504 N GLN A 859 48.545 30.765 34.866 1.00 62.94 N ATOM 2505 CA GLN A 859 49.881 31.342 34.974 1.00 65.25 C ATOM 2506 C GLN A 859 49.836 32.821 35.342 1.00 66.53 C ATOM 2507 O GLN A 859 49.409 33.659 34.547 1.00 67.06 O ATOM 2508 CB GLN A 859 50.649 31.146 33.665 1.00 65.62 C ATOM 2509 CG GLN A 859 50.925 29.689 33.340 1.00 66.74 C ATOM 2510 CD GLN A 859 51.310 29.478 31.890 1.00 67.56 C ATOM 2511 OE1 GLN A 859 52.220 30.128 31.373 1.00 68.15 O ATOM 2512 NE2 GLN A 859 50.616 28.560 31.224 1.00 67.40 N ATOM 2513 N GLN A 860 50.278 33.127 36.560 1.00 67.86 N ATOM 2514 CA GLN A 860 50.305 34.495 37.068 1.00 69.17 C ATOM 2515 C GLN A 860 51.602 35.221 36.709 1.00 69.76 C ATOM 2516 O GLN A 860 52.558 35.146 37.512 1.00 70.12 O ATOM 2517 CB GLN A 860 50.121 34.497 38.589 1.00 69.65 C ATOM 2518 CG GLN A 860 48.673 34.392 39.049 1.00 70.61 C ATOM 2519 CD GLN A 860 48.545 34.436 40.561 1.00 70.97 C ATOM 2520 OE1 GLN A 860 49.263 35.179 41.231 1.00 71.16 O ATOM 2521 NE2 GLN A 860 47.620 33.649 41.103 1.00 71.34 N ATOM 2522 OXT GLN A 860 51.653 35.845 35.626 1.00 70.21 O

Methods for predicting the effect on protein conformation of a change in protein sequence, are known in the art, and the skilled artisan can thus design a variant which functions as an antagonist according to known methods. One example of such a method is described by Dahiyat and Mayo in Science (1997) 278:82 87, which describes the design of proteins de novo. The method can be applied to a known protein to vary only a portion of the polypeptide sequence. Similarly, Blake (U.S. Pat. No. 5,565,325) teaches the use of known ligand structures to predict and synthesize variants with similar or modified function.

Other methods for preparing or identifying peptides that bind to a target are known in the art. Molecular imprinting, for instance, can be used for the de novo construction of macromolecular structures such as peptides that bind to a molecule. See, for example, Kenneth J. Shea, Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sites, TRIP Vol. 2, No. 5, May 1994; Mosbach, (1994) Trends in Biochem. Sci., 19 (9); and Wulff, G., in Polymeric Reagents and Catalysts (Ford, W. T., Ed.) ACS Symposium Series No. 308, pp 186-230, American Chemical Society (1986). One method for preparing mimics of a PDE5 inhibitor involves the steps of: (i) polymerization of functional monomers around a known substrate (the template) that exhibits a desired activity; (ii) removal of the template molecule; and then (iii) polymerization of a second class of monomers in, the void left by the template, to provide a new molecule which exhibits one or more desired properties which are similar to that of the template. Other binding molecules such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active materials can also be prepared. This method is useful for designing various biological mimics that are more stable than their natural counterparts, because they are prepared by the free radical polymerization of functional monomers, resulting in a compound with a nonbiodegradable backbone. Other methods for designing such molecules include, e.g., drug design based on structure activity relationships, which require the synthesis and evaluation of a number of compounds and molecular modeling.

The invention also provides in vivo and in vitro methods for identifying a compound that binds to a PDE5 protein. In one embodiment, the method comprises: (a) obtaining a tissue and/or cells that express the PDE5 protein; (b) contacting the tissue and/or cell with a ligand source for an effective period of time; (c) measuring a secondary messenger response, wherein the response is indicative of a ligand binding to PDE5 protein; (d) isolating the ligand from the ligand source; and (e) identifying the structure of the ligand that binds PDE5 protein, thereby identifying which compound would bind to PDE5 protein. As used herein, the term “ligand source” can be any compound library described herein, or a library of neurotransmitters that can be used to screen for compounds that would act as an agonist or antagonist of PDE5. Screening compound libraries listed herein [also see U.S. Patent Application Publication No. 2005/0009163, which is hereby incorporated by reference in its entirety], in combination with in vivo animal studies and functional and signaling assays can be used to identify PDE5 inhibitor compounds that can be used to treat subjects afflicted with abnormal A(3 deposits, such as AD.

A PDE5 inhibitor compound can be a compound that decreases the activity and/or expression of a PDE5 molecule in vivo and/or in vitro. PDE5 inhibitor compounds can be compounds that exert their effect on the activity of PDE5 via the expression, via post-translational modifications, or by other means. In one embodiment, a PDE5 inhibitor can decrease PDE5 protein or mRNA expression, or PDE5 activity by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, or 100%.

Test compounds or agents which bind to a PDE5 molecule, and/or have a stimulatory or inhibitory effect on the activity or the expression of a PDE5 molecule, can be identified by various assays. The assay can be a binding assay comprising direct or indirect measurement of the binding of a test compound or a known PDE5 ligand to the active site of a PDE5 protein. The assay can also be an activity assay comprising direct or indirect measurement of the activity of a PDE5 molecule. The assay can also be an expression assay comprising direct or indirect measurement of the expression of PDE5 mRNA or protein. The various screening assays can be combined with an in vivo assay comprising measuring the effect of the test compound on cognitive and synaptic function in an animal model for neurodegenerative disorders, such as AD. The activity of a PDE5 inhibitor can be measured in various ways, such as detecting an alteration in a downstream secondary messengers of the NO pathway [see FIG. 19]. The alteration can be in intracellular cyclic guanosine monophosphate (cGMP) concentration, in intracellular GTP concentration, in the intracellular protein kinase G (PKG) concentration, in the intracellular phosphorylation of CREB, or a combination thereof. For example, if an increase in cGMP levels is observed following administration of a PDE inhibitor and the inhibitor is detected or its metabolites in a dialysate, the test compound will be deemed active and thus a PDE5 inhibitor.

The diagnostic assay of the screening methods of the invention can also involve monitoring the expression of a PDE5 molecule. For example, inhibitors of the expression of a PDE5 molecule can be identified via contacting a PDE5-positive cell or tissue with a test compound and determining the expression of PDE5 protein or PDE5 mRNA in the cell. The protein or mRNA expression level of PDE5 in the presence of the test compound is compared to the protein or mRNA expression level of PDE5 in the absence of the test compound. The test compound can then be identified as an inhibitor of PDE5 expression based on this comparison. For example, when expression of PDE5 protein or mRNA is statistically or significantly less in the presence of the test compound than in its absence, the compound is identified as an inhibitor of the expression of PDE5 protein or mRNA. In other words, the test compound can also be said to be a PDE5 inhibitor compound (such as an antagonist). The expression level of PDE5 protein or mRNA in cells can be determined by methods described herein.

Determining the ability of a test compound to bind to a PDE5 molecule or a variant thereof, such as a PDE5 mutant described herein, can be accomplished using real-time Bimolecular Interaction Analysis (BIA) [McConnell, (1992); Sjolander, (1991)]. BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIA-core™). Changes in optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

Structure-Activity Relationship (SAR) of Known PDE5 Inhibitors

An analysis of the chemical structures of existing PDE5 inhibitors reveals that they can be divided into the following classes: 1) cGMP-based molecules, represented by sildenafil and vardenafil; 2) β-carbolines-derived molecules, represented by tadalafil; 3) pyrazolopyridine, phthalazine and quinoline derivatives; 4) isoquinazolinone and isoquinolinone derivatives.

As exemplified by sildenafil and vardenafil (Levitra, by Bayer), the cGMP-based PDE5 inhibitors are simple analogs of cGMP and differ only in the number and position of hetero-atoms such as nitrogen on the purine ring of cGMP (FIG. 43). These early PDE5 inhibitors show good potency but have poor selectivity over PDE1 and PDE6. For example, the IC₅₀ for PDE5 is 10 nM and 5 nM for sildenafil and vardenafil, respectively, but the PDE6/PDE5 selectivity ratios are only 12 for sildenafil and 3.5 for vardenafil [Boschelli et al., J Med Chem, 2001. 44 (5): p. 822-33; Wang et al., Bioorg Med Chem Lett, 2000. 10 (21): p. 2477-80]. Although sildenafil and vardenafil represent completely different classes of chemical structures based on the differences between their polycyclic cores, these two compounds share significant structural similarity, which explains the fact that both of them have poor selectivity. With the success of launching sildenafil and vardenafil, numerous efforts have been made to develop new PDE5 inhibitors based on pyrazolopryrimidinone core structure (for instance Udenafil by Dong-A Pharm). Unfortunately, most of the newly designed compounds inherited the poor selectivity over PDE6 from sildenafil even if they turned to be excellent PDE5 inhibitors. To improve the selectivity, several groups investigated modification on the phenyl ring of sildenafil. By changing the substitution pattern of the pyrazolo moiety of sildenafil combined with the transformation of phenyl to pyridine, selectivity was improved. This class of compounds also featured a nitrogen- or oxygen-containing substituent introduced on the nitrogen at the 6-position of the pyrazolopryrimidinone, which have high PDE5 potency and selectivity versus PDE6 up to 2482-fold [Barrios Sosa et al., Bioorg Med Chem Lett, 2004. 14 (9): p. 2155-8](see Pf-1 in FIG. 43, as well as, for example, WO 2002074774, WO 2002074312, EP 995750, WO 9849166, EP 995751, WO 9307149; EP 636626; U.S. Pat. No. 5,294,612, EP 1092718, WO 9924433; US 2003199693). It should be noted, however, that selectivity for remaining PDEs, as well as PK, BBB penetration, and toxicity profiles of this class of compounds are not known.

Molecules featuring β-carbolines constitute the basis for a 2nd class of PDE5 inhibitors. Ethyl beta-carboline-3-carboxylate (beta-CCE) was a weak, non-selective PDE5 inhibitor that led to the modestly selective hydantoin and, ultimately, to tadalafil (Clalis, by Lilly; FIG. 44). Tadalafil is a highly potent (IC₅₀=5 nM) and highly selective PDE5 inhibitor with selectivity of PDE5/PDE1-4 and PDE5/PDE6 over 1000, but poor selectivity for PDE11 (˜5) that can alter spermatogenesis and fertilization potential [Graham et al., Bioorg Med Chem Lett, 2007. 17 (21): p. 5886-93; Masliah, Histol Histopathol, 1995. 10 (2): p. 509-19]. If for an AD drug, fertility dysfunction is unlikely to represent a major problem, another side effect of tadalafil, back pain, can be a bigger problem for chronic use in a senile population [Selkoe et al., Science, 2002. 298 (5594): p. 789-91; Sant'Angelo et al., Neurochem Res, 2003. 28 (7): p. 1009-15; Bliss et al., Nature, 1993. 361 (6407): p. 31-9; Cullen et al., Neuroreport, 1997. 8 (15): p. 3213-7](incidentally, it is not clear whether this side effect is due to inhibition of PDE11 or other off target molecules). SAR studies have indicated that the NH group is essential. Alkylation of the nitrogen or replacement of nitrogen by sulfur abolishes activity is consistent with the role NH as an essential H-bond donor. SAR also indicated the hydrophobic aromatic ring (3,4-methylenedioxyphenyl) is necessary for high potency. PDE5 tolerates a wide range of substituents on the imide nitrogen of the hydantoin as well as the free piperazinedione nitrogen of tadalafil. At least one carbonyl group is important. Deletion of both markedly decreases potency, whereas removal of either one is only marginally deleterious [Wang et al., Bioorg Med Chem Lett, 2000. 10 (21): p. 2477-80]. Nonetheless, because of the poor selectivity problems related to PDE11 (which was not overcome), back pain, and its inability to cross the BBB (clogP=1.43; the NH group, which is quite acidic, is essential to its activity, but can make the compounds polar and consequently difficult to penetrate the BBB), it is unlikely that this compound can serve as a base for developing an AD drug.

A series of pyrimidinylpyrroloquinolones was also recently developed by Johnson & Johnson (JJ), as potent and selective PDE5 inhibitors. During the synthesis of JJ1 (FIG. 44), pyrroloquinolone JJ2 was formed as a minor byproduct. The potency of JJ2 against PDE5 exceeded the β-carboline and JJ1, because the high NH acidicity (pKa˜9) increases its propensity for hydrogen-bond with PDE5 ([Freir et al., J Neurophysiol, 2001. 85 (2): p. 708-13], WO 2001087882). Although JJ2 showed very good potency, superior selectivity and in vivo efficacy in a dog model for ED, poor solubility precluded further use of this compound. Similar to the SAR for tadalafil and analogs, a tolerance for a wide range of substituents on the pyrole nitrogen made it possible to develop desirable physical and chemical properties such as solubility and absorption while retaining potency and selectivity. As a consequence, JJ3 was developed and showed oral bioavailability of more than 30% in male rats, as well as good in vivo efficacy in a dog model of ED [Itoh et al., Eur J Pharmacol, 1999. 382 (3): p. 167-75; Kim et al., J Neurosci, 2001. 21 (4): p. 1327-33]. Additional features (full PDE selectivity profile, PK, BBB penetration) are not known.

In yet another series of structures, BMS1 was reported as a potent PDE5 inhibitor (IC₅₀=1 nM, FIG. 45). Using BMS1 as a template, Bristol-Myers Squibb (BMS) identified BMS2 (FIG. 3) as a PDE5 inhibitor with improved potency and selectivity compared to sildenafil (IC₅₀<0.8 nM) ((Stephan et al., J Neurosci, 2001. 21 (15): p. 5703-14), WO 2000015222). The X-ray structure of BMS2 showed that the benzylic amine —NH— formed a hydrogen bond with the amide carbonyl. This observation, coupled with the structure of Eisai's potent PDE5 inhibitor E1 (FIG. 45, IC₅₀=0.56 nM), EC₅₀=13 nM) (Vitolo et al., Proc Natl Acad Sci USA, 2002. 99 (20): p. 13217-21; WO 9807430), led the scientists at BMS to design compounds with constrained conformation and the pyrazolopyrido-pyridazine scaffold yielding the potent PDE5 inhibitor BMS3 (IC₅₀=0.3 nM, EC₅₀=13 nM, FIG. 45) with PDE1 and PDE6 isozyme selectivities superior to those of sildenafil. Of note, BMS3 had a desirable PK profile in two animal species with fewer PDE-related side effects such as visual disturbances (Walsh et al., Nature, 2002. 416 (6880): p. 535-9). More recently, BMS and a Japanese company independently reported that a combination of the important features of BMS2 and E1 led to a quinoline series of derivatives illustrated by BMS4. BMS4 is the most potent and selective PDE5 inhibitor to date, 30-fold more potent than sildenafil and significantly more selective than sildenafil against other PDE isozymes (IC₅₀=0.05 nM, >7800 selective versus PDE1-6) (Selig et al., Learn Mem, 1996. 3 (1): p. 42-8; WO 0112608, 2001). However, BMS4 lacks a complete PDE specificity profile, PK profile and in vivo efficacy against AD. In addition, the presence of benzylic alcohol causes concerns on off target side effects. Therefore, YF012403 has been developed as discussed herein.

Incorporation of an additional ring into cGMP-based PDE5 inhibitors generated a new class of structures. The fused 3-ring system, N-3 substituted imidazoquinazolinones, shows improved potency and selectivity compared to sildenafil (BMS5, IC₅₀=0.5 nM, PDE1-3/PDE5>10,000, PDE6/PDE5 60, FIG. 46). Incorporation of another nitrogen and a benzyl group into the middle ring forms another family of potent and selective PDE5 inhibitors represented by BMS6 (IC₅₀=0.31 nM, >10,000 fold selective vs. PDE1 and 160 fold vs. PDE6) (US 2002133008). Based on the reported in vitro properties, this scaffold may be of interest in terms of developing new PDE5 inhibitors because the value of PDE5/PDE6 reached 160, however, it needs to be pointed that the compounds derived from this scaffold may also cause some off-target toxicity since the IC₅₀ of BMS6 for PDE6 is around 50 nM, indicating that these derivatives may still be good PDE6 inhibitors.

The naphthalene analog TS1 was discovered by a Japanese company as a potent and selective PDE5 inhibitor (IC₅₀=6.2 nM, PDE1-4/PDE5>16000; FIG. 46). Superimposition of TS1 with cGMP shows that the naphthalene ring in TS1 significantly overlaps the purine nucleus in cGMP and the pendant phenyl group at the 1-position of TS1 fills a space occupied by the cyclic phosphate group in cGMP. Based on this observation, a class of potent and selective PDE5 inhibitors was identified as illustrated by T1032 (IC₅₀<1.0 nM, PDE1/PDE5, 1300, PDE2/PDE5>10 000, PDE3/PDE5>10 000, PDE4/PDE5 4700, PDE6/PDE5 28; FIG. 46). T1032 displays the most potent relaxant effect on isolated rabbit corpus cavernosum (EC₅₀ 7.9 nM) (Prickaerts et al., Eur J Pharmacol, 2002, 436 (1-2): p. 83-7; WO 9838168; JP 2000072675). Introduction of a nitrogen atom into the phenyl ring of T1032 led to yet another new structural class of potent and specific PDE5 inhibitors illustrated by T1056 (FIG. 46) with potent PDE5 inhibition (IC₅₀=0.23 nM) and excellent PDE5 selectivity against other PDEs1-4,6 (>100.000-fold selective versus PDE1-4, 240-fold selective vs. PDE6). This compound showed more potent relaxant effects on isolated rabbit corpus cavernosum (EC₅₀=5.0 nM) than sildenafil (EC₅₀=8.7 nM) [Ukita et al., Bioorg Med Chem Lett, 2003. 13 (14): p. 2341-5.]. However, since T1056 has an IC₅₀ of 56 nM against PDE6, it raises the same concern as BMS6.

Currently used AD therapies (acetylcholinesterase inhibitors or NMDA antagonists) have limited efficacy. Major efforts are underway to inhibit tangle formation, to combat inflammation and oxidative damage, and to decrease Aβ load in the brain either by the use of agents that inhibit β and γ secretases or increase secretase, by the use of drugs that inhibit Aβ oligomerization [Nakagami et al, Br J Pharmacol, 2002. 137 (5): p. 676-82; Walsh et al., J Neurosci, 2005. 25 (10): p. 2455-62], or by the use of treatments such as immunization with AP3 that appear to augment the removal of A from the brain [Schenk et al., Nature, 1999. 400 (6740): p. 173-7]. However, the role of APP, Aβ 40, and the secretases in normal physiological function [Wu et al., Eur J Pharmacol, 1995. 284 (3): p. R1-3; Kowalska et al., Biochem Biophys Res Commun, 1994. 205 (3): p. 1829-35; Mattson et al., J Neurochem, 1999. 73 (2): p. 532-7] can present a problem in providing effective and safe approaches to AD therapy.

Exemplary PDE5 Inhibitor Compounds Optimized for CNS Disorders

The invention provides for compounds that bind to PDE5. These compounds can be identified by the screening methods and assays described herein, and inhibit the activity or expression of PDE5 proteins. In one embodiment, the invention encompasses compounds of the following formulae:

wherein R¹, R², and R³ are each independently —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen.

In one embodiment, the invention encompasses compounds of Formula Ia:

wherein:

-   -   X is CR or N;     -   each R is independently —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl,         —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, —C₆-C₁₀         aryl, or halogen, at the 2, 3, or 4 position on the ring,         relative to X; and     -   R² is —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀         aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen, at the 2, 3, 4,         5, or 6 position on the ring.

In one embodiment, R is —H.

In another embodiment, R is —O—C₁-C₆ alkyl, such as —OCH₃.

In yet another embodiment, R is —C₁-C₆ alkyl-C₆-C₁₀ aryl, such as benzyl.

In one embodiment, the R of X and the R on the ring are different.

In another embodiment, R² is —H.

In one embodiment, the invention encompasses compounds of Formula Ib:

wherein:

-   -   X is CR or N;     -   R is —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀         aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen;     -   R¹ is —H, —C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, or —C₆-C₁₀         aryl; and     -   R² and R³ are each independently —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆         alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl,         or halogen.

In an embodiment, R is —H.

In one embodiment, the invention encompasses compounds of Formula Ic:

wherein:

-   -   X is CR, or N;     -   R is —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀         aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen;     -   R¹ is —H, —C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, or —C₆-C₁₀         aryl; and     -   R² and R³ are each independently —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆         alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl,         or halogen.

In an embodiment, R is —H.

In one embodiment, the invention encompasses compounds of Formula Id:

wherein:

-   -   X is CR, or N;     -   R is —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀         aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen;     -   R¹ is —H, —C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, or —C₆-C₁₀         aryl; and     -   R² and R³ are each independently —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆         alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl,         or halogen.

In an embodiment, R is —H.

In one embodiment, the invention encompasses compounds of Formula Ie:

wherein:

-   -   X is CR, or N;     -   R is —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀         aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen;     -   R¹ is —H, —C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, or —C₆-C₁₀         aryl;     -   R² and R³ are each independently —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆         alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl,         or halogen; and     -   R⁴ is —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀         aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, halogen, or —CO₂—C₁-C₆         alkyl.

In one embodiment, R is —H.

In another embodiment, R² is —OH.

In a further embodiment, R² is a halogen, such as —Cl.

In an embodiment, R³ is —H.

In another embodiment, R⁴ is —CO₂—C₁-C₆ alkyl, such as —CO₂Me.

In yet another embodiment, R⁴ is —H.

In one embodiment, the invention encompasses compounds of Formula IIa:

wherein:

-   -   R¹, R², and R³ are each independently —H, —OH, —C₁-C₆ alkyl,         —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl,         —O—C₆-C₁₀ aryl, or halogen.

In an embodiment, R² is aryl, such as phenyl.

In another embodiment, R¹ is —H.

In yet another embodiment, R³ is —H.

In one embodiment, the invention encompasses compounds of Formula IIb:

wherein:

-   -   R¹, R², and R³ are each independently —H, —OH, —C₁-C₆ alkyl,         —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl,         —O—C₆-C₁₀ aryl, or halogen.

In an embodiment, the C₆-C₁₀ aryl is substituted with one or more of —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen.

In an embodiment, R² is aryl, such as phenyl.

In another embodiment, R₂ is —C₆-C₁₀ aryl substituted with —C₁-C₆ alkyl, such as toluoyl.

In one embodiment, R¹ is —H.

In an embodiment, R³ is —H.

In another embodiment, R³ is —C₁-C₆ alkyl, such as isopropyl.

In one embodiment, the invention encompasses compounds of Formula IIc:

wherein:

-   -   R¹ and R³ are each independently —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆         alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl,         or halogen; and     -   R² is —H, —C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, or —C₆-C₁₀         aryl.

In an embodiment, R¹ is —H.

In one embodiment, R² is —H.

In another embodiment, R² is —C₁-C₆ alkyl-C₆-C₁₀ aryl, such as benzyl.

In still another embodiment, R³ is —H.

In one embodiment, the invention encompasses compounds of Formula IId:

wherein:

-   -   R² is —H, —C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C(O)—C₁-C₆         alkyl, or —C₆-C₁₀ aryl; and     -   R³ is —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀         aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen.

In one embodiment, C₆-C₁₀ aryl is substituted with one or more of —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen.

In an embodiment, R² is —C₁-C₆ alkyl-C₆-C₁₀ aryl, such as benzyl.

In another embodiment, R² is —C₁-C₆ alkyl-C₆-C₁₀ aryl, wherein the —C₆-C₁₀ aryl group is substituted with one or more of —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, or halogen.

In a specific embodiment, R² is —C₁-C₆ alkyl-C₆-C₁₀ aryl, wherein the —C₆-C₁₀ aryl group is substituted with —O—C₁-C₆ alkyl, such as —OMe.

In another specific embodiment, R² is —C₁-C₆ alkyl-C₆-C₁₀ aryl, wherein the —C₆-C₁₀ aryl group is substituted with halogen, such as —Cl.

In an embodiment, R³ is —H.

In a specific embodiment, R³ is halogen, such as —Cl.

In one embodiment, the invention encompasses compounds of Formula IIe:

wherein:

-   -   R¹ and R² are each independently —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆         alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl,         or halogen, and R¹ can be on the 5, 6, 7, or 8 position of the         quinoline ring; and     -   R³, R⁴, and R⁵ are each independently —H, —OH, —C₁-C₆ alkyl,         —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl,         —O—C₆-C₁₀ aryl, or halogen.

In one embodiment, C₆-C₁₀ aryl is substituted with one or more of —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen.

In an embodiment, R¹ is halogen, such as —Cl.

In one embodiment, the invention encompasses compounds of Formula IIIa:

wherein:

-   -   R is —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₁         aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen;     -   R¹ and R₂ are each independently —H, —C₁-C₆ alkyl, —C₁-C₆         alkyl-C₆-C₁₀ aryl, or —C₆-C₁₀ aryl.

In one embodiment, the invention encompasses compounds of Formula IIIa-1:

wherein:

-   -   R² is —H, —C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, or —C₆-C₁₀         aryl.

In one embodiment, the compound comprises Formula IIIb:

wherein:

-   -   R is —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀         aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen; and     -   R¹ and R² are each independently —H, —C₁-C₆ alkyl, —C₁-C₆         alkyl-C₆-C₁₀ aryl, or —C₆-C₁₀ aryl.

In an embodiment, R is —H.

In an embodiment, R¹ is —H.

In an embodiment, R² is —H.

In one embodiment, the compound of Formula IIIb is a compound of Formula IIIb-1:

wherein:

-   -   R¹ and R² are as defined for Formula IIIb.

In one embodiment, the invention encompasses compounds of Formula IIIc:

wherein:

-   -   R, R¹, and R² are each independently —H, —OH, —C₁-C₆ alkyl,         —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl,         —O—C₆-C₁₀ aryl, or halogen.

In one embodiment, R is —H.

In an embodiment, R¹ is halogen, such as —Cl.

In an embodiment, R² is halogen, such as —Cl.

In one embodiment, the invention encompasses compounds of Formula IIIc-1:

wherein:

-   -   R¹ and R² are as defined for Formula IIIa.

In one embodiment, the invention encompasses compounds of Formula IIId:

wherein:

-   -   R¹ is —H, —C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, or —C₆-C₁₀         aryl.

In one embodiment, the invention encompasses compounds of Formula IIIe:

wherein:

-   -   R¹ is —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀         aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl, or halogen; and     -   R² is —H, —C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, or —C₆-C₁₀         aryl.

In one embodiment, the invention encompasses compounds of Formula IIIf:

wherein:

-   -   R¹ and R² are each independently —H, —C₁-C₆ alkyl, —C₁-C₆         alkyl-C₆-C₁₀ aryl, or —C₆-C₁₀ aryl.

In one embodiment, the invention encompasses compounds of Formula IVa:

wherein:

-   -   R¹ and R³ are each independently —H, —OH, —C₁-C₆ alkyl, —O—C₁-C₆         alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl, —O—C₆-C₁₀ aryl,         or halogen; and     -   R² is —H, —C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, or —C₆-C₁₀         aryl.

In one embodiment, R¹ is —H.

In one embodiment, R² is —C₁-C₆ alkyl, such as methyl.

In one embodiment, R³ is —H.

In another embodiment, R³ is —C₁-C₆ alkyl or —C₁-C₆ alkyl-C₆-C₁₀ aryl.

In one embodiment, the invention encompasses compounds of Formula IVb:

wherein:

-   -   R¹, R², R³, R⁴, and R⁵ are each independently —H, —OH, —C₁-C₆         alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ alkyl-C₆-C₁₀ aryl, —C₆-C₁₀ aryl,         —O—C₆-C₁₀ aryl, or halogen.

In one embodiment, R¹ is —H.

In one embodiment, R² is —H.

In one embodiment, R³ is —H.

In one embodiment, R⁴ is —H.

In one embodiment, R⁵ is —H. In another embodiment, R⁵ is —C₁-C₆ alkyl or —C₁-C₆ alkyl-C₆-C₁₀ aryl.

In one embodiment, the compounds contain a fused planar ring system, and this ring system contains: (1) a hydrogen bond acceptor (e.g. N on pyrimidyl ring and C═O on sildenafil) or (2) an H-bond donor (NH) or H-bond acceptor (C═O) or both (amide NH—C═O).

In another embodiment, the compounds contain a fused planar ring system with 3 hydrophobic groups (R¹, R², and R³). The optimal size and nature of these 3 hydrophobic groups for tight binding to PDE5 seems to depend on the strength of hydrogen bonding between the enzyme and the H bond acceptor or donor. For inhibitors with a H bond acceptor (C═O, N:) on the fused planar ring system, a bulky aromatic R² group helps to achieve optimal fit at the site occupied by the phosphate of cGMP. For inhibitors with a H bond donor (i.e. NH of tadalafil) on the fused planar ring system, a bulky aromatic R¹ group helps to achieve optimal fit at the hydrophobic Q2 pocket. R³ can be small, and it appears to be less significant than R¹ and R². These observations comport with insights from the X-ray structures of the PDE5-inhibitor complexes. By modification of R¹, R², R³, the potency, selectivity and PK properties such as oral bioavailability, cellular penetration, and blood-brain barrier penetration can be fine-tuned.

In one embodiment, the invention encompasses compounds of Formula (V):

-   -   wherein:         -   A is O or N;         -   X is —(CH₂)_(n), C(O), S(O), or S(O)₂;         -   R¹ is hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, —NR⁷R⁸, —SR⁷,             or heterocyclyl;         -   R² is —CH₂OR⁶ or —CO₂R⁸;         -   R³ is hydrogen or halogen;         -   R⁴ is —CN or halogen;         -   R⁵ is hydrogen or —OR⁶;         -   R⁶ is hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹;         -   R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl,             —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or             C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆             alkyl, —C₃-C₈ cycloalkyl, —NR⁹R¹⁰, —SR⁹, or heterocyclyl;             or, R⁷ and R⁸ together with the nitrogen atom to which they             are attached form a 3 to 8-membered heterocycle, wherein any             one of the ring carbon atoms is optionally replaced with a             heteroatom, and wherein the heterocycle is optionally             substituted with C₁-C₆ alkyl; and         -   R⁹ and R¹⁰ are each independently hydrogen, C₁-C₆ alkyl, or             C₃-C₈ cycloalkyl; and n is 1, 2, or 3,         -   or a pharmaceutically acceptable salt or tautomer thereof.

In one embodiment, A is N.

In one embodiment, X is —(CH₂)_(n), where n is 1, 2, or 3.

In one embodiment, R¹ is hydrogen. In another embodiment, R¹ is cycloalkyl.

In one embodiment, R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷. In another embodiment, R¹ is C₃-C₈ cycloalkyl or —NR⁷R⁸. In still another embodiment, R¹ is C₃-C₈ cycloalkyl. In yet another embodiment, R¹ is —NR⁷R⁸. In still another embodiment, R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —NR⁹R¹⁰; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8-membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with O, NR⁹ or N—C(O)R⁹. In still another embodiment, R¹ is —SR⁷. In yet another embodiment, R¹ is —S—(C₁-C₆)—alkyl. In a specific embodiment, R¹ is cyclopropyl, while in another particular embodiment R¹ is dimethylamino.

In one embodiment, R² is CH₂—OH.

In one embodiment, R³ is H. In a specific embodiment, R³ is a halogen, such as chloro.

In one embodiment, R⁴ is —CN. In another embodiment, R⁴ is a halogen (for example, fluorine).

In one embodiment, R⁵ is hydrogen. In another embodiment, R⁵ is —OR⁶, where R⁶ is —C₁-C₆ alkyl, or —C₃-C₈ cycloalkyl. In a specific embodiment, R⁵ is —OCH₃.

In another embodiment, the compound is of formula (V-1):

-   -   wherein:         -   R¹ is hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, —NR⁷R⁸, —SR⁷,             or heterocyclyl;         -   R² is —CH₂OR⁶ or —CO₂R⁸;         -   R³ is hydrogen or halogen;         -   R⁴ is —CN or halogen;         -   R⁶ is hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹;         -   R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl,             —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or             C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆             alkyl, —C₃-C₈ cycloalkyl, —NR⁹R¹⁰, —SR⁹, or heterocyclyl;             or, R⁷ and R⁸ together with the nitrogen atom to which they             are attached form a 3 to 8-membered heterocycle, wherein any             one of the ring carbon atoms is optionally replaced with a             heteroatom, and wherein the heterocycle is optionally             substituted with C₁-C₆ alkyl; and         -   R⁹ and R¹⁰ are each independently hydrogen, C₁-C₆ alkyl, or             C₃-C₈ cycloalkyl,         -   or a pharmaceutically acceptable salt or tautomer thereof.

In one embodiment, R¹ is hydrogen. In another embodiment, R¹ is cycloalkyl.

In one embodiment, R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷. In another embodiment, R¹ is C₃-C₈ cycloalkyl or —NR⁷R⁸. In still another embodiment, R¹ is C₃-C₈ cycloalkyl. In yet another embodiment, R¹ is —NR⁷R⁸. In still another embodiment, R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —NR⁹R¹⁰; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8-membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with O, NR⁹ or N—C(O)R⁹. In still another embodiment, R¹ is —SR⁷. In yet another embodiment, R¹ is —S—(C₁-C₆)-alkyl. In a specific embodiment, R¹ is cyclopropyl, while in another particular embodiment R¹ is dimethylamino.

In one embodiment, R² is CH₂—OH.

In one embodiment, R³ is H. In a specific embodiment, R³ is a halogen, such as chloro.

In one embodiment, R⁴ is —CN. In another embodiment, R⁴ is a halogen (for example, fluorine).

In one embodiment, R⁶ is C.

In another embodiment, the compound is of formula (V-1a):

-   -   wherein:         -   R¹ is hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, —NR⁷R⁸, —SR,             or heterocyclyl;         -   R² is —CH₂OR⁶ or —CO₂R⁸;         -   R³ is hydrogen or halogen;         -   R⁴ is —CN or halogen;         -   R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl,             —C₃-C₈ cycloalkyl,         -   or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are             optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl,             —NR⁹R¹⁰, —SR⁹, or heterocyclyl or, R⁷ and R⁸ together with             the nitrogen atom to which they are attached form a 3 to             8-membered heterocycle, wherein any one of the ring carbon             atoms is optionally replaced with a heteroatom, and wherein             the heterocycle is optionally substituted with C₁-C₆ alkyl;             and         -   R⁹ and R¹⁰ are each independently hydrogen, C₁-C₆ alkyl, or             C₃-C₈ cycloalkyl,         -   or a pharmaceutically acceptable salt or tautomer thereof.

In one embodiment, R¹ is hydrogen. In another embodiment, R¹ is cycloalkyl.

In one embodiment, R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷. In another embodiment, R¹ is C₃-C₈ cycloalkyl or —NR⁷R⁸. In still another embodiment, R¹ is C₃-C₈ cycloalkyl. In yet another embodiment, R¹ is —NR⁷R⁸. In still another embodiment, R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —NR⁹R¹⁰; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8-membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with O, NR⁹ or N—C(O)R⁹. In still another embodiment, R¹ is —SR⁷. In yet another embodiment, R¹ is —S—(C₁-C₆)-alkyl. In a specific embodiment, R¹ is cyclopropyl, while in another particular embodiment R¹ is dimethylamino.

In one embodiment, R² is CH₂—OH.

In one embodiment, R³ is H. In a specific embodiment, R³ is a halogen, such as chloro.

In one embodiment, R⁴ is —CN. In another embodiment, R⁴ is a halogen (for example, fluorine).

In another embodiment, the compound is of formula (V-1a1):

-   -   wherein:         -   R¹ is hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, —NR⁷R⁸, —SR⁷,             or heterocyclyl;         -   R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl,             —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or             C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆             alkyl, —C₃-C₈ cycloalkyl, —NR⁹R¹⁰, —SR⁹, or heterocyclyl;             or, R⁷ and R⁸ together with the nitrogen atom to which they             are attached form a 3 to 8-membered heterocycle, wherein any             one of the ring carbon atoms is optionally replaced with a             heteroatom, and wherein the heterocycle is optionally             substituted with C₁-C₆ alkyl; and         -   R⁹ and R¹⁰ are each independently hydrogen, C₁-C₆ alkyl, or             C₃-C₈ cycloalkyl,         -   or a pharmaceutically acceptable salt or tautomer thereof.

In one embodiment, R¹ is hydrogen. In another embodiment, R¹ is cycloalkyl.

In one embodiment, R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷. In another embodiment, R¹ is C₃-C₈ cycloalkyl or —NR⁷R⁸. In still another embodiment, R¹ is C₃-C₈ cycloalkyl. In yet another embodiment, R¹ is —NR⁷R⁸. In still another embodiment, R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —NR⁹R¹⁰; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8-membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with O, NR⁹ or N—C(O)R⁹. In still another embodiment, R¹ is —SR⁷. In yet another embodiment, R¹ is —S—(C₁-C₆)-alkyl. In a specific embodiment, R¹ is cyclopropyl, while in another particular embodiment R¹ is dimethylamino.

In particular embodiments, the compound is:

In other embodiments, the compound is

In specific embodiments, the compound is

In one specific embodiment, the compound is:

In another specific embodiment, the compound is:

In one embodiment, the compounds of the invention do not include compounds of formula X:

wherein:

-   -   R², R⁶, R⁷ and R⁸ are independently hydrogen, halogen, alkyl,         substituted alkyl, alkoxy, nitro, cyano, aryl, heteroaryl, or         heterocyclo;     -   R³ is —(CH₂)_(z), Y, wherein z is 0, 1, 2, or 3;     -   R⁴ and R⁵ (i) are independently hydrogen, alkyl, substituted         alkyl, cycloalykl, substituted cycloalkyl, aryl, or heteroaryl,         with the proviso that R⁴ and R⁵ are not both hydrogen; (ii)         taken together form a heterocyclo ring; or (iii) one of R⁴ and         R⁵ together with Y forms a heterocyclo ring;     -   Y is (i) independently selected from —OR⁹, —CO₂R⁹, —CH(CO₂R⁹)₂,         —O(C═O)NR¹⁰R¹¹, —NR¹⁰R¹¹, —NR¹⁰(C═O)NR¹¹R¹², —CH[(C═O)NR¹⁰R¹¹]₂,         —(C═O)NR¹⁰R¹¹, —NR¹⁰(C═O)R¹², —S(O)_(m)R⁹, —SO₂NR¹⁰R¹¹,         imidazole, substituted imidazole, triazole, substituted         triazole, or cyano, or (ii) together with R⁴ or R⁵ forms a         heterocylo ring; and m is 0, 1, or 2;     -   R⁹ is hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy,         cycloalkyl, substituted cycloalkyl, heterocyclo, aryl,         heteroaryl, or pentafluorophenyl; and     -   R¹⁰, R¹¹, and R¹² are (i) independently selected from hydrogen,         alkyl, substituted alkyl, alkoxy, cycloalkyl, substituted         cycloalkyl, aryl, heterocyclo, and heteroaryl; or (ii) taken         together, wherein R¹⁰ forms a three-to seven-membered         heterocyclo ring with R¹¹ or R¹², or R¹¹ forms a three-to         seven-membered heterocyclo ring with R¹².

The invention also provides methods for increasing α-secretase protein activity or expression in a subject by administering any one of the compounds having Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ie, Formula IIa, Formula IIb, Formula IIc, Formula IId, Formula Ie, Formula IIIa, Formula IIIb, Formula IIIc, Formula IIIa-1, Formula IIIb-1, Formula IIIc-1, Formula IIId, Formula IIIe, Formula IIIf; Formula IVa, Formula IVb, Formula V, Formula V-I, Formula V-1-a, or Formula V-a-1 (such as any one of compounds 1-18) above. The invention also provides a method for decreasing β-secretase protein activity or expression in a subject by administering any one of the compounds having Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ie, Formula IIa, Formula IIb, Formula IIc, Formula IId, Formula IIe, Formula IIIa, Formula IIIb, Formula IIIc, Formula IIIa-1, Formula IIIb-1, Formula IIIc-1, Formula IIId, Formula IIIe, Formula IIIf; Formula IVa, Formula IVb, Formula V, Formula V-1, Formula V-1-a, or Formula V-a-1 (such as any one of compounds 1-18) above. In addition, the invention provides methods for reducing amyloid beta (AP) protein deposits in a subject by administering any one of the compounds having Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ie, Formula IIa, Formula IIb, Formula IIc, Formula IId, Formula IIe, Formula IIIa, Formula IIIb, Formula IIIc, Formula IIIa-1, Formula IIIb-1, Formula IIIc-1, Formula IIId, Formula IIIe, Formula IIIf; Formula IVa, Formula IVb, Formula V, Formula V-1, Formula V-1-a, or Formula V-a-1 (such as any one of compounds 1-18) above.

In some embodiments, compounds having Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ie, Formula IIa, Formula IIb, Formula IIc, Formula IId, Formula IIe, Formula IIIa, Formula IIIb, Formula IIIc, Formula IIIa-1, Formula IIIb-1, Formula IIIc-1, Formula IIId, Formula IIIe, Formula IIIf; Formula IVa, Formula IVb, Formula V, Formula V-1, Formula V-1-a, or Formula V-a-1 (such as any one of compounds 1-18) are first screened for their ability to satisfy one or more of the following characteristics: an IC₅₀ no greater than about 100 nM; a selectivity that is at least 50-fold greater for PDE5 than for other PDEs; a PDE5 inhibitory activity in vitro that has an IC₅₀ no greater than about 50 nM, the ability to penetrate the BBB; the ability to hydrolyze cGMP by at least about 20% (or at least about 80%); an interaction between the compound and PDE5 that comprises a second bridging ligand that is a hydroxyl group; and an interaction between the compound and PDE5 that comprises contacts with PDE5 at amino acid residues F787, L804, I813, M816, or a combination thereof. Thereafter or independently, the compounds can be tested for their ability to provide long-lasting effects on inhibiting β-secretase activity or expression and/or on activating α-secretase activity or expression (such as in the mouse APP transgenic model).

In one embodiment, the method comprises administering to the subject an effective amount of a composition comprising a PDE5 inhibitor compound. In another embodiment, the subject exhibits abnormally elevated amyloid beta plaques. In a further embodiment, the subject is afflicted with Alzheimer's disease, Lewy body dementia, inclusion body myositis, or cerebral amyloid angiopathy. In some embodiments, the Aβ protein deposit comprises an Aβ₄₀ isomer, an Aβ₄₂ isomer, or a combination thereof. In further embodiments, α-secretase protein activity or expression is increased up to 3 months post-treatment, up to 4 months post-treatment, up to 5 months post-treatment, or up to 6 months post-treatment. In other embodiments, β-secretase protein activity or expression is decreased up to 3 months post-treatment, up to 4 months post-treatment, up to 5 months post-treatment, or up to 6 months post-treatment.

PDE5 inhibitor compounds of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions can comprise a PDE5 inhibitor compound (such as sildenafil, tadalafil, vardenafil, or a compound comprising Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ie, Formula IIa, Formula IIb, Formula IIc, Formula IId, Formula IIe, Formula IIIa, Formula IIIb, Formula IIIc, Formula IIIa-1, Formula IIIb-1, Formula IIIc-1, Formula IIId, Formula IIIe, Formula IIIf; Formula IVa, Formula IVb, Formula V, Formula V-1, Formula V-1-a, or Formula V-a-1 (such as any one of compounds 1-18) and a pharmaceutically acceptable carrier. The compositions can be administered alone or in combination with at least one other agent, such as a stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones. In one embodiment, the effective amount of a PDE5 inhibitor compound can be at least about 3 mg/kg body weight. In another embodiment, the composition is administered at least once daily for up to 18 days, up to 19 days, up to 20 days, up to 21 days, up to 22 days, up to 23 days, up to 24 days, or up to 25 days.

Non-limiting examples of additional PDE5 inhibitors include: 5-[2-ethoxy-5-(4-methyl-1-piperazinylsulphonyl)phenyl]-1-methyl-3-n-propy-1-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (sildenafil) also known as 1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulphonyl]-4-methylpiperazine (see EP-A-0463756); 5-(2-ethoxy-5-morpholinoacetylphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see EP-A-0526004); 3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-n-propoxyphenyl]-2-(pyridin-2-yl)methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO98/49166); 3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-methoxyethoxy)pyridin-3-yl]-2-(pyridin-2-yl)methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO99/54333); 6-benzo[1,3]dioxol-5-yl-2-methyl-2,3,6,7,12,12a-hexahydro-pyrazino[1′,2′:-1,6]pyrido[3,4-b]indole-1,4-dione (cialis); (+)-3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-methoxy-[(R)-methyl-1 ethoxy)pyridin-3-yl]-2-methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, also known as 3-ethyl-5-{5-[4-ethylpiperazin-1-ylsulphonyl]-2-([(1R)-2-methoxy-1-methyl-ethyl]oxy)pyridin-3-yl}-2-methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-1-one (see WO99/54333); 5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, also known as 1-{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazol-o[4,3-d]pyrimidin-5-yl]-3-pyridylsulphonyl}-4-ethylpiperazine (see WO01/27113, Example 8); 5-[2-iso-butoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-(1-methylpiperidin-4-yl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO01/27113, Example 15); 5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-phenyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO01/27113, Example 66); 5-(5-acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidin-yl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO01/27112, Example 124); 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO01/27112, Example 132); (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2′,1′:6,1]pyrido[3,4-b]indole-1,4-dione (IC-351), i.e. the compound of examples 78 and 95 of published international application WO95/19978, as well as the compound of examples 1, 3, 7 and 8; 2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one (vardenafil) also known as 1-[[3-(3,4-dihydro-5-methyl-4-oxo-7-propylimidazo[5,1-f]-as-triazin-2-yl)-4-ethoxyphenyl]sulphonyl]-4-ethylpiperazine, i.e. the compound of examples 20, 19, 337 and 336 of published international application WO99/24433; the compound of example 11 in WO93/07124 (EISAI); and compounds 3 and 14 from Rotella D P, J. Med. Chem., 2000, 43, 1257.

According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.

Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the PDE5 inhibitor compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

EXAMPLES

Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

Example 1 Sildenafil (Viagra) Leads to an Immediate and Persistent Improvement of Hippocampal Synaptic Plasticity, Memory and AβLoad in an Alzheimer Mouse Model

This example discusses whether sildenafil can exert beneficial effects against synaptic dysfunction and memory loss of mice carrying both the mutant amyloid precursor protein (APP; K670N,M671L) and presenilin-1 (PS1; M146L) (termed APP/PS1 mice). The PDE5 inhibitor sildenafil (Viagra) was tested to see whether it was beneficial against the AD phenotype in a mouse model of amyloid deposition. The inhibitor produces an immediate and long-lasting amelioration of synaptic function, CREB phosphorylation and memory. This effect was associated with a reduction of Aβ levels. Thus, PDE5 inhibitors have potential for the treatment of AD and other diseases associated with elevated Aβlevels.

Previous studies show that a) NO protects against Aβ-induced LTP block; b) sGC is involved in NO-protection against Aβ-induced LTP block; c) cGMP and activation of its downstream target, PKG, have a beneficial effect against Aβ-induced LTP block; d) increase in NO and cGMP levels can protect against Aβ suppression of phospho-CREB increase during LTP; e) sGC is involved in NO protection against Aβ-induced block of increase in phospho-CREB during LTP; f) PKG is involved in the effect of cGMP analogs on CREB phosphorylation during Aβ treatment; g) Aβ-induced suppression of LTP is associated with block of the increase in cGMP levels (Puzzo, D., et al., J Neurosci, 2005. 25 (29): p. 6887-97). Various explanations support these findings: a) given that the cofactor NADPH strongly complexes soluble Aβ, which results in its diminished availability for NOS functioning [Colton, C. A., et al., Proc Natl Acad Sci USA, 2006. 103 (34): p. 12867-72], Aβ can function as a sink for NADPH, preventing the production of NO by NOS and consequently halting the resulting cascade of events that includes cGMP production and ends with CREB phosphorylation; b) an increase of PDE activity following Aβ application [Wirtz-Brugger, F. and A. Giovanni, Neuroscience, 2000. 99 (4): p. 737-50]; c and d) a decrease of sGC activity and/or expression [Baltrons, M. A., et al., Neurobiol Dis, 2002. 10 (2): p. 139-49](i.e. PDE activity increase has been demonstrated on both isolated blood vessels and cultured microglia in which PDE5 inhibition re-establishes normal vasoactivity and blocks inflammatory response due to Aβ [Paris, D., et al., Exp Neurol, 1999. 157 (1): p. 211-21]); and f) a reduction of NOS expression. Although identifying the link between the NO cascade and Aβ-induced synaptic dysfunction would constitute an important research subject, new PDE5 inhibitor drugs will be developed.

Acute effects of sildenafil on synaptic function in hippocampal slices of app/psi mice. A brief application of sildenafil was tested to see whether it rescued the defect in LTP of slices derived from 3 month-old APP/PS1 mice, when synaptic plasticity impairment is just starting whereas basal synaptic transmission (BST) is normal^(A12). BST was determined by measuring the slope of the field excitatory postsynaptic potential (fEPSPs) at increasing stimulus intensity in APP/PS1 and wild-type (WT) mice. No difference in BST among the different groups was observed (FIG. 1a ). Hippocampal slices were then perfused with sildenafil (50 nM) for 10 min before inducing LTP through tetanic stimulation of the Schaeffer collateral pathway. Potentiation in sildenafil treated APP/PS1 slices was far greater than in vehicle-treated APP/PS1 slices (FIG. 1b ). On the other hand, sildenafil did not change the amplitude of LTP in slices of WT mice compared to WT slices treated with vehicle alone (FIG. 1c ). Sildenafil had no effect on basal synaptic responses either during its application or 120 minutes after the end of the application in experiments where no tetanus was applied either in slices from APP/PS1 mice or WT littermates (FIG. 1b,c ).

As a control for PDE5 specificity of the sildenafil effect onto synaptic dysfunction, a more specific PDE5 inhibitor, tadalafil, was used. Differently than sildenafil and vardenafil which are cGMP based inhibitors, tadalafil is a β-carbolines-derived drug with no effect on PDE1 (selectivity ratio>2000) and on PDE6 (selectivity ratio 1000), and an IC₅₀ against PDE5 of 5.0 nM^(A11). In these experiments slices were bathed in 50 nM tadalafil for 10 min prior to tetanus. Potentiation in tadalafil-treated APP/PS1 slices was far greater than in vehicle-treated APP/PS1 slices (FIGS. 8A-B). Tadalafil did not change the amplitude of baseline and LTP in WT mice (FIGS. 8A-B).

As an additional control for PDE5 specificity, IC354 was also used a PDE1 inhibitor. It is the HCl salt of IC224^(A13), a highly selective PDE1 inhibitor (IC₅₀ against PDE1 of 80 nM; ratio of IC₅₀ value for the next most sensitive PDE to IC₅₀ value for PDE1 equal to 127). In these experiments slices were bathed in 1 μM IC354 for 10 min prior to tetanus. Differently than sildenafil or tadalafil, the treatment did not augment LTP. Potentiation in IC354 treated APP/PS1 slices was similar to vehicle-treated APP/PS1 slices (FIGS. 8C-D) and IC354 did not change the amplitude of LTP in hippocampal slices of WT mice (FIGS. 8C-D). Thus, these results taken together with the experiments with sildenafil and tadalafil demonstrate that inhibition of PDE5 (but not PDE1) protects AD-like animal models against synaptic dysfunction, supporting that inhibition of PDE5 can be beneficial against synaptic dysfunction in AD.

Acute effects of sildenafil on the cognitive function of app/psi mice. Given that sildenafil reversed LTP deficits in 3-month-old APP/PS1, it was then tested whether the inhibitor reversed the cognitive defects in these animals. As reported above sildenafil offers the advantage of crossing the BBB and therefore it can be easily utilized in behavioural experiments. Three-month-old mice were divided into 4 groups: APP/PS1 with sildenafil, APP/PS1 with vehicle, WT with sildenafil and WT with vehicle. Sildenafil and vehicle control solutions were administered i.p. at a concentration of 3 mg/kg. This concentration was chosen based on previous studies showing that these amounts of sildenafil raise hippocampal cGMP levels and improve memory in aged rats^(A6) and mice^(A7) independent of vascular effects^(A6). The effects of acute administration of sildenafil was first examined on fear-conditioning learning, a type of learning that is impaired in several AD mouse models^(A14), and depends on hippocampus and amygdala. The hippocampus is indispensable for contextual fear conditioning^(A15), a form of associative learning in which mice must associate a neutral stimulus with an aversive one. Mice were trained to associate neutral stimuli with an aversive one. They were placed in a new context (fear conditioning box), exposed to a white noise cue (CS) paired with a mild foot shock (US), and injected with sildenafil immediately after the training. Fear learning was assessed twenty-four hours later by measuring freezing behaviour—the absence of all movement except for that necessitated by breathing—in response to representation of the context or of the auditory cue within a completely different context. No difference was found in the freezing behaviour among the four groups of mice before the training phase (FIG. 2a ). Twenty-four hours later, a decrease in the freezing behaviour of vehicle-treated APP/PS1 mice compared with that of vehicle-treated WT littermates in the analysis of the contextual learning was observed (FIG. 2a ). Sildenafil treatment improved contextual learning in the transgenic animals (FIG. 2a ) whereas sildenafil-treated WT animals did not show a significant increase in freezing (FIG. 2a ), probably because maximal levels of memory are already induced in vehicle-treated WT mice after the training session, as has been found both in Drosophila and in mice^(A16, A17). Fear conditioning, a hippocampus-independent task^(A15), was next tested and no difference in freezing among the 4 groups was found, as APP/PS1 mice are known to have a selective hippocampus-dependent impairment in associative learning^(A14).

To exclude the possibility that sildenafil produced its behavioural effect through a peripheral vascular action, the study of fear memory was repeated using tadalafil which is unable to cross the BBB (cLogP=1.43 and information from its manufacturer). Tadalafil and vehicle control solutions were administered i.p. at a concentration of 1 mg/kg. Tadalafil did not improve associative learning in APP/PS1 mice. Thus, the effect of sildenafil cannot be due to inhibition of PDE5 in the vascular compartment (FIG. 9).

Next, the effect of treatment with sildenafil was examined on spatial working memory, a type of short-term memory that can be studied with the radial-arm water maze test. This task has already demonstrated memory deficits in other transgenic models of AD^(A12, A18) and has been shown to depend upon hippocampal function^(A19). Mice were required to learn and memorize the location of a hidden platform in one of the arms of a maze with respect to spatial cues. APP/PS1 injected with vehicle showed severe abnormalities in spatial memory for platform location during both acquisition and retention of the task compared to vehicle-injected WT littermates (FIG. 2b ). However, daily injections of sildenafil for 3 weeks immediately after the 4^(th) acquisition trial ameliorated the behavioural performance of APP/PS1 mice (FIG. 2b ). Treatment with sildenafil did not affect the performance of WT mice compared to vehicle-injected WT littermates (FIG. 2b ). The four groups of mice showed no difference in the time needed to find the platform in the visible platform task, as well as in swimming speed (FIG. 10). Thus, vision, motor coordination, or motivation were not affected in the four groups of mice and did not influence the radial-arm water maze test results.

Persistent effects of sildenafil on cognitive and synaptic functions in app/psi mice. Sildenafil was tested to determine whether a brief course of treatment can provide long term benefits. The PDE5 inhibitor was examined to see if it maintains its protective effect against synaptic dysfunction and memory loss. In these experiments, both APP/PS1 and WT mice of 3 months of age were injected intraperitoneally with 3 mg/kg/day sildenafil for 3 weeks, then the treatment was stopped for 9-12 weeks prior to testing. The mice were next subjected to training for contextual learning. As in the acute experiments, when the animals were reintroduced into the same context in which they had been trained 9-12 weeks before, the freezing time was greatly increased in APP/PS1 mice that had been previously treated with sildenafil compared to vehicle-treated APP/PS1 littermates (FIG. 3a ). Sildenafil did not increase the freezing time in WT littermates compared to WT mice treated with vehicle (FIG. 3a ). There were no differences between the 4 groups in the cued conditioning test. These data indicate that inhibition of PDE5 protects fear contextual learning in APP/PS1 mice for an extended time beyond the duration of drug administration.

The effects of one course of 3-week treatment with sildenafil on spatial working memory were next tested using the radial-arm water-maze task. There was a difference between the number of errors made by vehicle-treated APP/PS1 and WT mice (FIG. 3b )^(A12). Administration of sildenafil for 3 weeks, 9-12 weeks prior to the testing, reduced the gap between the two groups without affecting performance of the WT animals (FIG. 3b ). These data indicate that one course of long-term treatment with the PDE5 inhibitor protects spatial working memory in APP/PS1 mice.

To investigate sildenafil effect on long-term memory, reference memory was tested with a Morris water maze task that is known to require hippocampal function^(A20) and is impaired after 6 months of age in the APP/PS1 mice^(A12). Vehicle-treated transgenic mice needed more time to find the hidden platform after six sessions compared to WT littermates (FIG. 3c ). When APP/PS1 mice were treated previously with sildenafil they showed a marked improvement of their behavioural performance. Sildenafil did not affect the performance in WT littermates (FIG. 3c ). Reference memory was also assessed with the probe trial, another test of spatial reference memory^(A20). This task is performed after the sixth hidden-platform session. The platform is removed from the water and the animals are allowed to search for 60 seconds. A mouse, knowing that the platform was in a certain position, will trawl repeatedly over that position looking for it. The mouse is thus indicating that it knows the position independently of such tactile cues as hitting the platform. The amount of time spent in each quadrant of the maze can be used to evaluate the spatial bias of an animal's search pattern. Vehicle-treated WT mice spent more time in the target quadrant (TQ), where the platform had been located during training than in other quadrants, than in the adjacent quadrant to the right (AR), in the adjacent quadrant to the left (AL), or in the opposite quadrant (OQ) (FIG. 3d ). Also, sildenafil improved the performance of the APP/PS1 mice which searched more in the quadrant where the platform had been located during training than in other quadrants (FIG. 3d ). In contrast, vehicle-treated APP/PS1 mice did not retain the information and spent less time in the TQ compared to vehicle-treated WT littermates. Sildenafil-treated WT mice remembered where the platform was the previous days and spent about the same time as vehicle-treated WT littermates. A visible platform trial performed after the probe trials did not reveal any difference in the time to reach the platform and swimming speed among the 4 groups (FIGS. 11A-B).

To add depth to the analysis of the functional changes that underlie the striking effects of sildenafil on APP/PS1 mice behavioral performance, synaptic function in hippocampi from the same mice was examined. In contrast to 3-month-old double transgenic mice, 8- to 9-month old APP/PS1 animals show a reduction of synaptic strength^(A12). Previous treatment with sildenafil in APP/PS1 mice produced greater values of fEPSP slope in response to a 35V stimulus in slices from 8 to 9 month old then in vehicle-treated APP/PS1 littermates slices (FIG. 4a ). On the other hand, sildenafil did not change responses in WT littermates. CA3-CA1 connections that had been tested for BST were also assessed for their capacity of undergoing potentiation. LTP values recorded from slices obtained from APP/PS1 that had been previously treated with sildenafil were similar to their sildenafil treated-WT littermates and far greater than those from vehicle-treated APP/PS1 littermates (FIG. 4b,c ). Eight-to nine-month old WT mice showed similar amounts of potentiation whether treated with sildenafil or with vehicle (FIG. 4c ). No differences were noted in the baseline transmission of the four groups of mice in the absence of tetanus (FIG. 4b,c ). Taken together, these data indicate that one course of treatment with sildenafil is protects APP/PS1 mice against synaptic dysfunction for a long time.

Effects of sildenafil on creb phosphorylation in app/psi mice. Given that the duration of action of sildenafil is relatively short, a direct effect of the PDE5 inhibitor cannot be held responsible for its long-term effects. CREB has been implicated in the regulation of genes whose expression results in the formation and stabilization of long-term memory and CREB phosphorylation is required for CREB ability to bind to CREB binding protein (CBP) and to stimulate CRE dependent gene expression^(A21). Aβ elevation is also known to block the tetanus-induced increase in phosphorylation of the memory molecule CREB (Puzzo, D., et al. Amyloid-beta peptide inhibits activation of the nitric oxide/cGMP/cAMP-responsive element-binding protein pathway during hippocampal synaptic plasticity. J Neurosci 25, 6887-6897 (2005))^(A22). Thus, to gain insights into the mechanism by which the inhibitor produces the long-term changes in synaptic physiology and behaviour, levels of CREB phosphorylation were measured in sildenafil- and vehicle-treated transgenic and WT mice. Hippocampal slices were treated as described in the electrophysiological experiments, fixed 60 minutes after the treatment, stained with anti-phospho-CREB antibodies at Ser-133, and viewed on a confocal microscope. An increase in the intensity of immunofluorescence in CA1 cell body area of WT mice after application of the tetanus compared to control non-tetanized slices (FIG. 5a,b ) was observed. APP/PS1 animals did not have the physiological increase of CA1 phospho-CREB immunofluorescence after tetanus (FIG. 5a,b ), however, sildenafil re-established normal phospho-CREB increase in tetanized slices of the double transgenics (FIG. 5a,b ). Sildenafil did not affect the tetanus-induced increase in immunofluorescence in WT animals (FIG. 5a,b ).

Mice were injected with 3 mg/kg/day sildenafil or vehicle at the age of 3 months and then left without treatment for 9-12 weeks. An increase in immunofluorescence intensity in CA1 cell body area of WT mice after application of the tetanus compared to non-tetanized control slices was observed (FIG. 5c ). APP/PS1 mice did not reveal the physiological increase of phospho-CREB after tetanus but previous treatment with sildenafil re-established it (FIG. 5c ). Moreover, phospho-CREB immunofluorescence did not vary in slices from sildenafil-treated WT mice with tetanic stimulation (FIG. 5c ). Thus, without being bound by theory, at the root of the long-term improvement in synaptic physiology and behaviour there is the re-establishment of the increase of CREB phosphorylation in APP/PS1 mice following tetanic stimulation of the Schaffer collateral-CA1 connection.

Effects of sildenafil on Aβ levels of app/psi mice. Improvement in CREB phosphorylation in the APP/PS1 mice was examined with respect to whether the inhibitor also affected Aβ levels, a hallmark of AD. ELISA of extracts of cerebral cortices revealed a difference in human Aβ₄₀ and Aβ₄₂ levels both immediately after 1 course of 3 week treatment with sildenafil at the age of 3 months in APP/PS1 mice and in mice that were sacrificed after the second round of behavioural testing at 7-10 months (FIGS. 6a,7a ). Thus, without being bound by theory, a reduction in Aβ levels is at the bases of the prolonged beneficial effect by sildenafil. Aβ originates from APP through a proteolytic process catalyzed by secretases, producing different fragments with characteristic functions^(A24). APP is first cleaved by α and β secretases that generate soluble extracellular fragments, named α-APPs and β-APPs, and three forms of carboxyterminal fragments (CTFs): C83 by α-secretase cleavage, C89 and C99 by β-secretase cleavage^(A25, A26). CTFs are substrates of γ-secretase generating C-terminal peptides of 57-58 residues (APP intracellular domain, AID) and Aβ fragments from CTFβ^(A27). To assess whether the decrease in Aβ levels was related to changes in APP processing [see FIG. 20], western blot analyses on mice brain cortex for full length APP and its fragments was performed. No differences in levels of full-length APP and sAPPα were observed in 3 months old APP/PS1 mice treated with vehicle or sildenafil (FIG. 6 b,c,d), whereas a decrease in sAPPβ (FIG. 6b,e ) and an increase of the CTF fragment C83 and C99 were found in APP/PS1 after sildenafil treatment compared to vehicle-treated transgenics (FIG. 6b,f ).

APP levels were found unchanged also in 7-10 months old transgenic mice (FIG. 7b,c ). They have been treated with daily injections of sildenafil for 3 weeks at age of 3 months. APP/PS1 mice treated with sildenafil showed an increase of sAPPα protein levels, showing that the treatment modifies α-secretase activity (FIG. 7b,d ). Moreover, a decrease in sAPPβ in transgenic mice treated with sildenafil was observed (FIG. 7b,e ) showing a down-regulation of BACE activity. Analysis of the levels of the CTF fragments C83 and C99 did not reveal any change due to the treatment (FIG. 7b,f ). Thus, without being bound by theory, the reduction in Aβ levels by sildenafil is due to an action of the inhibitor onto α- and β-secretase activity.

Discussion

It is shown that a treatment with the PDE5 inhibitor sildenafil rescues synaptic and memory deficits in a transgenic mouse model of amyloid deposition. Sildenafil also re-establishes the increase in phosphorylation of the transcription factor and memory molecule CREB. In addition, the inhibitor counteracts the negative effects of high levels of Aβ on synaptic function, memory and CREB phosphorylation not only immediately, but also for a prolonged period beyond the drug administration. Finally, sildenafil causes an immediate and long-lasting reduction in Aβ₄₀ and Aβ₄₂ levels. Sildenafil causes a prolonged reduction in Aβ levels which in turn re-establishes normal synaptic function and memory.

A relevant finding of the present study is the beneficial effect of sildenafil onto synaptic dysfunction in a mouse model of amyloid deposition. This finding is consistent with studies on slices showing that cGMP increase through the use of NO donors or cGMP analogs rescues the reduction of LTP and the inhibition of CREB phosphorylation induced by exogenous application of Aβ (Puzzo, D., et al. J Neurosci 25, 6887-6897 (2005)). Given that altered synaptic function is a fundamental aspect in the cognitive decline of AD^(A28), an advantage of using PDE5 inhibitors in AD can be that this class of compounds will counteract aspects of the disease linked to synaptic dysfunction that can be relevant to memory loss.

Another discovery reported in the study is the reversal of the memory impairment in an amyloid-depositing mouse model following PDE5 inhibition. These results are in agreement with the observation that NO-mimetic molecules can reverse the cognitive impairment caused by scopolamine^(A29), or by forebrain cholinergic depletion^(A30), showing that stimulating the NO/cGMP signal transduction system can provide new, effective treatments for cognitive disorders. With regard to the beneficial effect on memory, it is interesting to note that inhibition of PDE5 activity during a narrow time window immediately after training for fear learning or after acquisition of the spatial task (but not 5 min before training for fear learning or acquisition of the spatial task) improves learning in the transgenic animals. Considering that the in vivo half-life of sildenafil is 0.4 hrs in rodents^(A10), there can be a time-window during the first 20-25 min after the electric shock or the acquisition of the spatial task during which learning processes are susceptible of improvement by PDE5 inhibition. Moreover, given that the beneficial effect of sildenafil was observed with its injection after the training, inhibition of PDE5 acts on memory consolidation mechanisms, and not on aspects of performance, such as perception of pain or of the environment.

In the present studies a brief course of sildenafil was still beneficial after 3 to 5 months from the drug administration. Considering that sildenafil has a short half-life, this effect can be due to a long-lasting synaptic modification through an action on gene expression. CREB has been implicated in the regulation of genes whose expression results in the formation and stabilization of long-term memory probably through the formation of new synaptic connections^(A16). When phospho-CREB binds to CREB binding protein (CBP), it stimulates CRE dependent gene expression. CBP functions as a co-activator that facilitates interactions with the basal transcription machinery by working as an acetyltransferase that catalyzes acetylation of the histone H3 of the chromatin, causing a loss in chromosomal repression and increase in the transcription of memory associated genes. Histone acetylation can be self-perpetuating, creating a functionally stable chromatin state and thus chronic changes in the rates of specific gene expression^(A31-A33). Thus, without being bound by theory, the prolonged beneficial effect of sildenafil is due to a permanent increase in histone acetylation. Inhibition of histone de-acetylation that is normally due to a group of enzymes with a reverse effect of CBP, re-establishes normal LTP and memory in APP/PS1 mice^(A34).

Decrease in Aβ levels by PDE5 inhibition in transgenic mice is another important finding of the present studies. Without being bound by theory, the beneficial effect of sildenafil is specific to PDE5 inhibition because tadalafil, a highly selective PDE5 inhibitor reproduced the effect of sildenafil on synaptic dysfunction, whereas IC354, a selective inhibitor of PDE1, another PDE that can be inhibited by sildenafil, did not re-establish normal LTP in slices from the double transgenic mice. Moreover, differently than rolipram which did not improve spatial working memory immediately after its administration, sildenafil immediately augmented spatial working memory. Most importantly, a striking difference between the effect of sildenafil and those of rolipram is that the former reduced Aβ levels in the brains of APP/PS1 mice, whereas the latter did not affect Aβ concentration.

When proposing a new class of drugs as therapeutic agents it is important to consider their side effects. This can have determined the failure of PDE4 inhibitors to enhance memory. An advantage of using PDE5 inhibitors is that their side effects are known as they have already been utilized for many years. Priapism has been reported to occur in a few cases following the intake of PDE5 inhibitors. However, the current view about the cause of priapism is that it is due to a dysregulation of PDE5 function following down-regulation of the NO pathway^(A40)—a phenomenon that is also caused by Aβ increase (Puzzo, D., et al. Amyloid-beta peptide inhibits activation of the nitric oxide/cGMP/cAMP-responsive element-binding protein pathway during hippocampal synaptic plasticity. J Neurosci 25, 6887-6897 (2005))—such that, paradoxically, PDE5 inhibitors have been proposed as therapeutic agents against priapism^(A41, A42). Additional adverse events of the PDE5 inhibitors include mild vasodilatory effects such as headache, flushing, dyspepsia, and nasal congestion or rhinitis, which can warrant caution in proposing PDE5 inhibitors as AD agents. However, although Aβ is primarily accumulating in the CNS, Aβ is also present in the blood of patients affected by AD and other neurological disorders characterized by abnormal Aβ production^(A43, A44). Intriguingly, systemic Aβ potentiates vasoconstriction not only in cerebral vasculature but also in other districts of the vascular system^(A45-A51). Moreover, hypertension is often associated with AD^(A46, A48, A49). Thus, it is very appealing to think that PDE5 inhibitors can counteract not only memory loss and Aβ generation, but also vascular symptoms that often affect AD patients

Drugs acting on the NO-cascade have vascular effects that can affect the cognitive performance. Thus, an alternative explanation for the beneficial effect of sildenafil is that the inhibitor works through a vascular effect instead of an intra-neuronal effect. This is unlikely as inhibition of PDE5 re-established normal LTP in slices directly exposed to PDE5 inhibitors. Moreover, although cAMP analogues have been shown to induce more dilatation of cerebral arterioles in the parietal cortex than cGMP analogues^(A55), only 8-Br-cGMP (but not 8-Br-cAMP) improved memory performance in rodents^(A6) showing that vascular mechanisms can not account for the cGMP effects. Most importantly, tadalafil that does not cross the BBB did not reproduce the behavioral effects of sildenafil.

The present findings are in agreement with reports showing that upregulation of the NO cascade has a protective effect on Aβ-induced damage in the CNS^(A56-A58). For instance, studies performed on PC12 cells, sympathetic neurons and hippocampal neurons, have shown that treatment with the NO generator S-nitroso penicillamine has a neuroprotective action through nitrosylation that inhibits the pro-apoptotic factor caspase-2^(A57). Aβ has been found to impair NO generation by different mechanisms including a decrease in NMDA receptor signal transduction^(A56), subtraction of NADPH availability to NOS^(A59), and inhibition of the phosphorylation of the serine-threonine kinase Akt^(A51). The superior temporal cortex of AD patients shows a reduction in soluble guanylyl cyclase activity^(A60). Soluble guanylyl cyclase is decreased following Aβ exposure in brain astroglial cells^(A61). PDE activity increase has been found on both isolated blood cells and cultured microglia, in which PDE5 inhibition re-establishes normal vasoactivity and blocks inflammatory response caused by Aβ^(A62). However, NO has also been viewed as a major agent of neuropathology and cell death when it is produced in high quantity. High amounts of NO lead to generation of significant quantity of peroxinitrites that are responsible for oxidative and nitrosative stress in Aβ-induced cell death^(A63-A69). These opposite findings can be reconciled with the findings with the observation that release of low amounts of NO by the constitutive forms of NOS including both the neuronal and the endothelial isoforms, n-NOS and e-NOS, promotes synaptic plasticity and learning, whereas uncontrolled production of high amounts of the gas by the inducible form of NO-synthase (iNOS) can promote oxidative and nitrosative stress via production of peroxinitrite^(A63-A69) [see FIG. 19 and FIG. 21]. The current status of drug research exploiting these discoveries is focused both on finding ways to upregulate the NO cascade and therefore elicit neuroprotection, as well as on finding ways to block peroxinitrite toxic effects in order to limit neuropathology^(A70). The present therapeutic strategy intervening with PDE5 offers the advantage of bypassing NO production by focusing on steps at the downstream level of NO generation [see FIG. 19 and FIG. 21].

Conclusion

Sildenafil treatment ameliorates synaptic and cognitive dysfunction in AD mouse mode. Agents increasing cGMP levels rescue the reduction of L-LTP, Tetanus-induced increase of CREB phosphorylation, and Contextual learning in APP/PS1 mice. The beneficial effect of the increase in cGMP levels by sildenafil on cognition, synaptic transmission and CREB phosphorylation can be extended beyond the duration of its administration.

Methods

Animals: Double transgenic mice expressing both the human APP (K670M:N671L) and PS1 (M146L) (line 6.2) mutations were compared to WT littermates. They were obtained by crossing APP with PS1 animals. To identify the genotype of the animals, the polymerase chain reaction was used on samples of the tail^(A12). All experiments were performed using male mice. The animals were maintained on a 12-12 h light-dark cycle (with light onset at 06:00 hours) in temperature and humidity controlled rooms of the Columbia University Animal Facility. Food and water were available ad libitum.

Drug preparation: Sildenafil was synthesized in 6 steps according to reported procedures (Terrett et al., 1996) (U.S. Pat. No. 5,346,901. 1994). Briefly, commercially available 2-ethoxybenzoic acid was converted to 2-ethoxybenzoyl chloride with thionyl chloride. Reaction of 2-ethoxybenzoyl chloride with 4-amino-1-methyl-3-N-propylpyrazole-5-carboxamide yielded the amide in 90% yield. Cyclization of the amide using NaOH afforded pyrazolopyrimidinone in 77% yield. Chlorosulfonylation of the pyrazolopyrimidinone in chlorosulfonic acid, followed by reaction with N-methylpiperazine provided sildenafil in 90% yield. Tadalafil was also synthesized according to reported procedures (Daugan et al., 2003b). Briefly, D-tryptophan methyl ester reacted with piperonal under Pictet-Spengler reaction condition (TFA/CH2Cl2/MeOH) and the resulting product condensed with chloroacetyl chloride to provide acylated intermediate. Reaction of the intermediate with N-methyl amine provided tadalafil. Tadalafil was diluted in 0.1% DMSO.

Drug administration: Three-month-old APP/PS1 and WT mice were separated into 4 groups: APP/PS1 mice treated with vehicle, APP/PS1 mice treated with PDE inhibitor, WT mice treated with vehicle, and WT mice treated with PDE inhibitor. In the experiments assessing the acute effects of PDE inhibition on synaptic dysfunction sildenafil (50 nM) or tadalafil (50 nM) or IC354 (1 μM) were directly given to the hippocampal slices through the perfusion system for 10 min prior to the theta burst [see FIG. 23]. In the remaining experiments, sildenafil was injected via i.p. The drug was administered either acutely or for 1 course of 3 week treatment. For assessment of the short-term effects of sildenafil, the drug was given at a concentration of 3 mg/kg immediately after the training. This dose yields concentrations of ˜2.5 μM cGMP in the hippocampus^(A53). For assessment of long-term effects, sildenafil was given daily by i.p. injection at a concentration of 3 mg/kg for 3 weeks and then treatment was stopped for 9-12 weeks prior to behavioral testing. Contextual and cued fear conditioning was performed for 3 days. Radial-arm water-maze (RAWM) was performed for 3 weeks. Then, the animals were sacrificed for electrophysiological recordings. To decide the time of administration of sildenafil in the acute experiments, a series of studies was performed in which the inhibitor was injected i.p. at 5 min before the electric shock or at 5 min before the first acquisition trial with the radial arm water maze. No beneficial effect was observed both on the freezing time and the number of errors in sildenafil-injected APP/PS1 mice (sildenafil-treated APP/PS1 mice demonstrated a freezing time equal to ˜90% that of vehicle-treated APP/PS1 mice; n=7 males for sildenafil-treated transgenics and 6 males for vehicle-treated transgenics, P>0.05; ˜5 errors in the retention trial for both sildenafil- and vehicle-treated transgenics, n=6 males for sildenafil-treated transgenics and 5 males for vehicle-treated transgenics, P>0.05, sildenafil did not affect the behavioral performance of WT mice in both tasks, n=5 males for all the conditions). Thus, all the behavioral experiments on the acute effects of sildenafil reported in the result section were performed with injection after the training.

Electrophysiological analysis: Animals were sacrificed by cervical dislocation followed by decapitation. Hippocampi were quickly removed. Transverse hippocampal slices (400 μm) were cut and recorded according to standard procedures^(A54). For example, following cutting hippocampal slices were transferred to a recording chamber where they were maintained at 29° C. and perfused with artificial cerebrospinal fluid (ACSF) continuously bubbled with 95% O₂ and 5% CO₂. The ACSF composition in mM was: 124.0 NaCl, 4.4 KCl, 1.0 Na₂HPO₄, 25.0 NaHCO₃, 2.0 CaCl₂, 2.0 MgSO₄, 10.0 glucose. CA1 fEPSPs were recorded by placing both the stimulating and the recording electrodes in CA1 stratum radiatum. BST was assayed either by plotting the peak amplitude of the fiber volley against the slope of the fEPSP, or by plotting the stimulus voltages against slopes of fEPSP. For LTP experiments, a 15 min baseline was recorded every min at an intensity that evokes a response ˜35% of the maximum evoked response. LTP was induced using θ-burst stimulation (4 pulses at 100 Hz, with the bursts repeated at 5 Hz and each tetanus including 3 ten-burst trains separated by 15 sec). In a set of experiments LTP was induced with 1 or 2 ten-burst trains to assess the effect of sildenafil on LTP induced with a different strength of the tetanus. Responses were recorded for 2 hrs after tetanization and measured as field-excitatory-post-synaptic potential (fEPSP) slope expressed as percentage of baseline. The results were expressed as mean±Standard Error Mean (SEM).

Behavioral studies—fear conditioning: This type of cognitive test is much faster than other behavioral tasks that require multiple days of training and testing^(A14, A17). The conditioning chamber was in a sound-attenuating box. A clear Plexiglas window allowed the experimenter to film the mouse performance with a camera placed on a tripod and connected to the Freezeframe software (MED Ass. Inc.). To provide background white noise (72 dB), a single computer fan was installed in one of the sides of the sound-attenuating chamber. The conditioning chamber had a 36-bar insulated shock grid floor. The floor was removable, and after each experimental subject, the floor was cleaned with 75% ethanol and then with water. Only one animal at a time was present in the experimentation room. For the cued and contextual conditioning experiments, mice were placed in the conditioning chamber for 2 min before the onset of a discrete tone (CS) (a sound that lasted 30 sec at 2800 Hz and 85 dB). In the last 2 sec of the CS, mice were given a foot shock (US) of 0.50 mA for 2 sec through the bars of the floor. After the CS/US pairing, the mice were left in the conditioning chamber for another 30 sec and were then placed back in their home cages. Freezing behavior, defined as the absence of all movement except for that necessitated by breathing, was scored using the Freezeview software.

To evaluate contextual fear learning, freezing was measured for 5 min (consecutive) in the chamber in which the mice was trained 24 hr after training. To evaluate cued fear learning, following contextual testing, the mice were placed in a new context (triangular cage with smooth flat floor and with vanilla odorant) for 2 min (pre-CS test), after which they were exposed to the CS for 3 min (CS test), and freezing will be measured. Sensory perception of the shock was determined through threshold assessment. A sequence of single foot shocks was delivered to animals placed on the same electrified grid used for fear conditioning. Initially, a 0.1 mV shock was delivered for 1 sec, and the animal behavior was evaluated for flinching, jumping, and vocalization. At 30 sec intervals the shock intensity was increased by 0.1 mV to 0.7 mV and then returned to 0 mV in 0.1 mV increments at 30 sec intervals. Threshold to vocalization, flinching, and then jumping was quantified for each animal by averaging the shock intensity at which each animal manifests a behavioral response to the foot shock. No difference was observed among different groups of mice in the experiments in which fear conditioning was tested in the presence of sildenafil or vehicle.

Behavioral studies—spatial working memory: This is a type of short-term memory that can be studied with the RAWM test¹². Briefly, the RAWM consisted of a white tank (120 cm diameter) filled with water (24-25° C.) and made opaque by the addition of non-toxic white paint. Within the tank walls were positioned so as to produce six arms, radiating from a central area. Spatial cues were presented on the walls of the testing room. At the end of one of the arms was positioned a clear 10 cm submerged (1.5 cm) platform that remained in the same location for every trial in one day, but was moved about randomly from day to day. On each trial the mouse started the task from a different randomly chosen arm. The mouse did not use its long-term memory of the location of the platform on previous days, but relied on the short-term memory of its location on the day in question based on spatial cues that were present in the room. Each trial lasted 1 min and errors were counted each time the mouse entered the wrong arm or needed more than 20 sec to reach the platform. After each error the mouse was pulled back to the start arm for that trial. After 4 consecutive acquisition trials, the mouse was placed in its home cage for 30 min, then returned to the maze and administered a 5^(th) retention trial. Testing was considered completed as the WT mice made the same number of errors at the 4^(th) and 5^(th) trial. The scores for each mouse on the last three days of testing were averaged and used for statistical analysis.

Behavioral studies—reference memory: The task studied with the Morris water maze has been previously described¹². Briefly, mice were trained in 2 daily sessions (4 hours apart), each consisting of 3 trials (1 minute each), for 3 days. Time required to reach the hidden platform was recorded. To test the retention of the spatial memory, 4 probe trials were performed after the training with the platform moved. The maze was divided into 4 quadrants. The percent of time spent in the quadrant that previously contained the platform was recorded and analyzed with a video tracking system (HVS 2020; HVS Image).

Behavioral studies—visible platform testing: Visible-platform training to test visual and motor deficits was performed in the same pool as in the Morris water maze, with the platform marked with a black flag and positioned randomly from trial to trial. Time to reach the platform and speed was recorded and analyzed with a video-tracking system (HVS 2020, HVS Image, UK).

Immunocytochemical experiments: Immunocytochemical measurements of phosho-CREB were performed as previously described (Puzzo, D., et al. Amyloid-beta peptide inhibits activation of the nitric oxide/cGMP/cAMP-responsive element-binding protein pathway during hippocampal synaptic plasticity. J Neurosci 25, 6887-6897 (2005)). Briefly, hippocampal slices were fixed in ice-cold 4% paraformaldehyde at 1 minute after the treatment. Slices were washed three times in phosphate-buffered saline (PBS), treated with 0.3% Triton X-100 for 60 minutes, washed three times in PBS again, treated with 50 mM ammonium chloride for 20 minutes and incubated in 10% goat serum for 60 minutes. Slices were incubated with the primary antibody (rabbit polyclonal anti-phospho-CREB from Upstate Biotechnology diluted 1:100 in 10% goat serum) for 36 hours at 4° C. Slices were washed in PBS (6 times, 2 hours each time), incubated with the secondary antibody (goat anti-rabbit antibody labelled with Alexa Fluor 488, from Molecular Probes), diluted 1:100 in 10% goat serum, for 12 hours at 4° C. and washed in PBS again (6 times, 2 hours each time). Slices were examined by confocal microscopy (Nikon D-Eclipse C1) using a 4× and a 16× objective. Kalman averages of 4 scans were collected for each image. The analysis was performed using the NIH image software by an observer who was blind to the experimental treatment. The mean fluorescence intensity that exceeded a threshold set above background was determined for each slice in CA1 cell body area. The values were normalized to the values from untreated control slices from the same animal and expressed as mean percent of control±SEM. The specificity of the immunofluorescence was confirmed by omitting the primary antibody, which resulted in a significant reduction in fluorescence intensity.

Determination of Aβ levels: Frozen hemi-brains were weighed and homogenized in 5 M guanidine HCL/50 mM Tris HCL solution. Aβ₄₀ and Aβ₄₂ were measured using human β amyloid ELISA kits (Biosource, CA), according to the manufacturer's protocol. ELISA signals were reported as the mean±s.e.m. in nanograms of Aβ per milligram of cortex. For example, hippocampal slices were fixed in ice-cold 4% paraformaldehyde at 1 minute after the treatment. Slices were washed three times in phosphate-buffered saline (PBS), treated with 0.3% Triton X-100 for 60 minutes, washed three times in PBS again, treated with 50 mM ammonium chloride for 20 minutes and incubated in 10% goat serum for 60 minutes. Slices were incubated with the primary antibody (rabbit polyclonal anti-phospho-CREB from Upstate Biotechnology diluted 1:100 in 10% goat serum) for 36 hours at 4° C. Slices were washed in PBS (6 times, 2 hours each time), incubated with the secondary antibody (goat anti-rabbit antibody labelled with Alexa Fluor 488, from Molecular Probes), diluted 1:100 in 10% goat serum, for 12 hours at 4° C. and washed in PBS again (6 times, 2 hours each time). Slices were examined by confocal microscopy (Nikon D-Eclipse Cl) using a 4× and a 16× objective. Kalman averages of 4 scans were collected for each image. The analysis was performed using the NIH image software by an observer who was blind to the experimental treatment. The mean fluorescence intensity that exceeded a threshold set above background was determined for each slice in CA1 cell body area. The values were normalized to the values from untreated control slices from the same animal and expressed as mean percent of control±SEM. The specificity of the immunofluorescence was confirmed by omitting the primary antibody, which resulted in a significant reduction in fluorescence intensity.

Western blot: Mice brains were homogenized in buffer (20 mM tris base, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose) containing protease inhibitors. Part of the homogenates was ultracentrifugated at 100,000 g for 1 h and the supernatants were used to analyze the sAPPα and the sAPPβ fragments. The protein concentration in each homogenate was quantified to analyze the equal amounts of protein from each sample. Samples were loaded into the wells of a 4-12% Bis-Tris precast gel, electrophoresed and transferred according to manufacturer's protocol. After Ponceau S staining, membranes were washed and incubated in 5% non-fat milk powder in PBS for 1 h at room temperature. Then, they were incubated overnight at 4° C. with the following primary antibodies, diluted in goat serum 5% in PBS: mouse 22C11 for APP full length detection (1:4000, Sigma); mouse sAPPα and Swedish sAPPβ (1:4000 and 1:1000, IBL), rabbit AbD for CTFs (1:250, Zymed), mouse Tubulin as control (1:20.000, Sigma). After overnight incubation, membranes were washed 3 times in PBS for 30 minutes and incubated with goat anti-mouse or goat-anti-rabbit antibody, diluted in 5% non-fat milk powder in PBS (1:4000 and 1:2000). After 30 minutes washing in PBS, radiographic detection was performed after exposure to ECL (Pierce). The analysis was performed using the NIH image software by an observer who was blind to the experimental treatment. The mean intensity that was determined for each samples and the values were normalized to the values from tubulin and expressed as mean percent of control±SEM.

Statistical analyses: For all experiments mice were coded to “blind” investigators with respect to genotype and treatment. Data were expressed as mean±SEM. Results were analyzed with student t-test (pairwise comparisons) or ANOVA with post-hoc correction (multiple comparisons). The level of significance was set for P<0.05.

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Example 2 Identification of PDE5 Inhibitors which are Optimized for AD—Compounds with High Affinity for PDE5 and Good Selectivity Relative to Other PDEs

Synaptic transmission and cognition are altered in double Tg (transgenic) mice expressing both the human amyloid precursor protein (APP) mutation (K670M:N671L) and the human presenilin-1 (PS1) mutation (M146L), termed APP/PS1 mice [23]. None of the existing PDE5 inhibitors has been developed to counteract diseases of the CNS and at the same time possesses the selectivity required for chronic administration to an elderly population with comorbid conditions such as AD patients. PDE5 inhibitors that are tailored to be used in AD patients can be screened, and can be tested to see whether these compounds can re-establish normal cognition in Tg AD models.

Enhancement of the NO/sGC/cGMP/PKG/CREB pathway through inhibition of PDE5 counteracts Aβ-induced synaptic and cognitive abnormalities. Drugs that both act on the NO/sGC/cGMP/PKG/CREB pathway and are optimized for the CNS are lacking. New drugs will be identified with a) high specificity and potency, b) good PK, bioavailability and CNS penetration, c) safety. None of the existing drugs is known to fit all of these criteria. Sildenafil is reported to cross the BBB [72] and has an IC₅₀ against PDE5 of 6.0 nM and an in vivo half-life of 0.4 hrs in rodents (˜4 hrs in humans) [70,74]. However, the selectivity ratio for PDE1, which is expressed in myocardium and blood vessels besides the brain and can result in mild vasodilatatory effects is 180, and that for PDE6, which is expressed only in retina and can transiently disturb vision is equal to 12 [67, 86]. Evidence for Vardenafil ability to cross the BBB is indirect [87](IC₅₀ against PDE5 0.17 nM, selectivity ratio for PDE1>1000 and PDE6=3.5) [88,89], and Tadalafil does not cross it (tadalafil also does not improve cognitive performance in APP/PS1 mice) (IC₅₀ against PDE5 5 nM, selectivity ratio for PDE1>2000 and PDE6=1000) [86]. A Structure-Activity Relationship (SAR) analysis of reported PDE5 inhibitors will be conducted and then a Computer-Aided MedChem Strategy will be employed to develop compounds that can fit all of these criteria.

Medicinal chemistry strategy. The strength of this proposal lies in the intensive use of functional assays at every stage of the project and the parallel use of a validated in vivo disease model. The medicinal chemistry approach must be tailored to suit the strengths of the bioassays and the reality that the resources for medicinal chemistry are an order of magnitude lower than can be found in industry. Existing PDE5 inhibitors are used as treatment of ED. Based on the structure analysis of reported PDE5 inhibitors and known SAR data (FIG. 12), four class of structurally related, but nevertheless formally independent scaffolds I-IV (see FIG. 13), are deemed as PDE5 inhibitor candidates. Compounds derived from these scaffolds will be screened and optimized on the computational models. Compounds with highest score will be synthesized and for potency. At this stage, the synthetic effort will be guided by the testing results of potency/selectivity. Compounds with satisfactory potency and selectivity (lead compounds) will be further studied for PK, bioavailability/brain penetration and off-target activities (safety).

Structure-Activity Relationship (SAR) of known PDE5 inhibitors: Although the design of early PDE5 inhibitors relied on mimicking the structure of cGMP, now a broad array of SAR data and very recent high resolution X-ray structures of PDE5 complexed with sildenafil, vardenafil and tadalafil are available and will provide a great basis to develop PDE5 inhibitors with the desired properties. After a survey of multiple distinct scaffold structures for PDE5 inhibitors and careful analysis of the SAR data from published reports, the scaffolds share a very common and important feature: all structures contain a fused planar ring system, and this ring system contains: (1) a hydrogen bond acceptor (e.g. N on pyrimidyl ring and C═O on sildenafil) or (2) an H-bond donor (NH) or H-bond acceptor (C═O) or both (amide NH—C═O). These observations comport with insights from the X-ray structures of the PDE5-inhibitor complexes. In addition to this planar ring system, all of the PDE5 inhibitors contain 3 hydrophobic groups (R¹, R², R³). The size and nature of these 3 hydrophobic groups seems to depend on the strength of H bonding between the enzyme and the H bond acceptor or donor. For inhibitors with a H bond acceptor (C═O, N:) on the fused planar ring system, a bulky aromatic R² group achieves optimal fit at the site occupied by the phosphate of cGMP. For inhibitors with a H bond donor (i.e. NH of tadalafil) on the ring system, a bulky aromatic R1 group achieves optimal fit at the hydrophobic Q2 pocket. R³ seems to be small and less significant compared with R¹ and R². By modification of R¹, R², R³, the potency, selectivity and other PK properties such as oral bioavailability and cellular penetration can be optimized. The fused planar ring systems in thus far reported PDE5 inhibitors are listed in FIG. 12.

Scaffolds to be synthesized: Based on the SAR analysis above, four sets of scaffolds (FIG. 13) are presented: 1) cGMP-based, represented by sildenafil (Viagra) and vardenafil (Levitra) (Pfizer, Bayer, Sheering-Plough), 2) β-carbolines-derived, represented by tadalafil (Clalis) (Lilly, Johnson&Johnson (J&J), 3) quinazoline and isoquinolinone derivatives (Bristol-Myers-Squibb (BMS), Japan), 4) phthalazine derivatives (BMS, Japan). These compound classes meet the following criteria: 1) a fused ring system with an H-bond acceptor or donor; 2) readily synthesized from readily available starting materials; 3) sufficient sites modifiable to generate a relatively large number of compounds for screening.

The design of scaffolds Ia-Ic is based on the known structures T1056 (shown in FIG. 13) with potent PDE5 inhibition (IC₅₀=0.23 nM) and excellent PDE5 selectivity against other PDEs1-4,6 (>100.000-fold selectivity versus PDE1-4, 240-fold vs. PDE6) (WO 9838168; JP 2000072675). In scaffold Ia, R² hydrophobic groups, to fit the site of phosphate of cGMP, can be introduced readily by Suzuki cross-coupling reaction with a versatile intermediate 5 (scheme Ia). If another R¹ group is required for Q2 pocket, the NH of the amide can be the site for substitution. In scaffold Ib, the enamine moiety is replaced with an amide. In scaffold Ic, the S═O will function as an H-bond acceptor.

Scaffolds were also based on the quinoline structure (IIa-IId) listed in FIG. 13. Two patents report quinoline derivatives as PDE5 inhibitors with general structure IIe (WO 2001012608, JP 2002308877). The quinoline-based PDE5 inhibitors reported by BMS is 120-fold more potent than sildenafil and significantly more selective than sildenafil against other PDE isozymes (IC₅₀=0.05 nM, PDE1-4/PDE5>7800, and PDE6/PDE5=160). From this compound, new compounds will be developed. For example the nitrogen can be replaced by O, C or S, or hetero atom can be introduced at various positions of either ring to generate new classes of compounds. Little SAR information is available on the quinolines, but a series of molecules, which can be synthesized in 2-3 steps and are generally isosteric with structure IIe, would guide predicted derivatives for in silico screening (see synthesis of scaffold IIa-IIc).

Scaffold IIIa-c and IIIa-1-IIIc-1 illustrated in FIG. 13 are designed based mainly on the observation that all reported PDE5 inhibitors possess a planar fused ring system with a H-bond acceptor or donor. Hydrophobic interactions are the predominant force in tadalafil binding, and related scaffolds are illustrated by IVa and IVb (FIG. 13).

Synthesis of scaffold 1a: The synthesis of target compound Ia is outlined in Scheme Ia (FIG. 14). The key intermediate quinolinone 3 can be obtained by cyclization of malonamide 2, which can be readily prepared from malonyl dichloride and substituted anilines, in the presence of commercially available Eaton's reagent. Quinolinone 3 is then converted to the 4-chloro derivative 5 through a straightforward two-step chlorination/hydrolysis sequence. Suzuki cross-coupling reaction allows compound 5 to couple with arylboronic acid, yielding the target compound Ia in good yield

Synthesis of scaffold IIa-IIc: IIc will be synthesized by reaction of substituted aniline with ethoxymethylenemalonic ester at high temperature, followed by an alkylating reactant such as benzyl bromide, which would yield IIc-4. The intermediate 4-hydroxyquinoline (IIc-1) can be converted to 4-chloroquinoline IIc-2, a very versatile intermediate. The reaction of IIc-2 with different amines provides large number of compounds, e.g. 4-aminoquinoline IIc-3 can be obtained from 4-chloroquinoline by reaction with ammonia. IIc-3 can be readily converted to amide IIa-1 and sulphonamide IIb-1 (Scheme IIa-IIc; FIG. 15).

Synthesis of scaffold IId: IId can be synthesized as outlined in scheme IId (FIG. 16). Anthranilic acid is treated with excess formamidine acetate at high temperature to yield compound IId-1. The chloride product IId-2 is obtained by treatment of IId-1 with thionyl chloride at reflux. Coupling chloride IId-2 with 3-chloro-4-methoxybenzylamine provides IId-3.

Synthesis of scaffold III: (see Scheme III; FIG. 17) Reaction of 2-aminobenzoic acid methyl ester with dimethyl acetylenedicarboxylate followed by a cyclization induced by t-BuOK provides product IIIc-1. Treatment of IIIc-1 with NH₂NH₂ yields the product IIIb-1 which is converted to IIIa-1. Reaction of IIIa-1 with amine provides IIIa. Reaction of IIIc-1 with amine provides IIIc. Alkylation of IIIb-1 provides IIIb.

Synthesis of scaffold IVa: As shown in Scheme IVa (FIG. 18), the target compounds IV can be prepared from the readily available amino acid methyl ester. Reaction with aromatic aldehydes provides the imine IVa-1. 1,3-dipolar cycloaddition of imine IVa-1 with naphthoquinone yields the key intermediate IVa-2. The target piperazinedione IVa can be obtained by acetylation of compound IVa-2, followed by ring closure in the presence of primary amines.

Evaluation of Drug Activity

In vitro tests: Candidate compounds will be tested for PDE5 inhibitory activity first. If the activity is modest, the compounds will be tested against other PDEs to evaluate selectivity. The PDEs will be purchased or prepared according to the methods described in the literature. A PDE assay will be performed according to reported methods [90,91] using Multiscreen plates (Millipore) and a vacuum manifold (Millipore), available in the lab, on which both the reaction and the subsequent separation of substrates and products can be achieved. The assay will use 50 mM Tris pH 7.5, 5 mM Mg acetate, 1 mM EGTA, and 250 μg/mL snake venom nucleotidase, 50 nM [8-³H]-cGMP (15 Ci/mmol; Amersham) or [8⁻³H]-cAMP (25 Ci/mmol; Amersham). Reactions are started by the addition of 25 μL of the diluted enzyme preparation. The assays will be incubated for 30 min at 30° C. Microcolumns will be prepared by aliquoting 300 μL per well of QAE Sephadex previously swollen for 2 hrs in water (12 mL/g). At the end of the incubation, the total volume of the assays will be loaded on microcolumn plate by filtration. The elution of free radioactivity will be obtained by 200 μL of water from which 50 μL aliquots will be analyzed by scintillation counting.

In addition to being potent inhibitors of PDE5, the candidate compounds must also be selective (some of the side-effects by known inhibitors are believed to be due, at least in part, to non-specific inhibition of other PDEs, such as PDE1 being found in heart and PDE6 primarily located in retina; see also [92] for a review). When assayed against other PDE families, they must show at least a 50 fold greater potency towards PDE5. These families include PDE1, Ca2+/calmodulin dependent; PDE2, cGMP-stimulated; PDE3, cGMP-inhibited; PDE4, cAMP-specific; PDE5, cGMP-specific; PDE6, photoreceptor cGMP-specific; PDE7, cAMP-specific; PDE8, cAMP-specific; PDE9, cGMP-specific; PDE10 and PDE11, hydrolyzing both cAMP and cGMP. In addition, these compounds have to be selective against kinases, such as PKG, PKA, PKC and PKB. Assays for these PDEs, as well as PKG and PKA, are well-established in the lab.

Tests in primary cultures and adult mice: Compounds with sufficient PDE5 inhibitory activity in vitro (IC₅₀<50 nM) will be further tested in a functional assay to determine whether the compounds can increase cGMP in the neurons. Hippocampal neurons will be prepared as previously described [93] and seeded in 24-well culture dishes at a density of (1-2)×10⁵ cells/well. Experiments will be performed after 10 days in culture when cells will reach confluence and form synaptic contacts. Media will be aspirated and replaced with 0.5 mL of PBS containing the PDE inhibitor. After 30 min at 37° C., soluble guanylyl cyclase will be stimulated by addition of 100 μM BAY 41-2272, a sGC stimulator with no effects on PDE activity [94], for 10 min at 37° C. At the end of the incubation, the medium will be removed and stored at −20° C. for extracellular cyclic nucleotides determinations. Intracellular cyclic nucleotides will be extracted by two ethanolic (65%) washes at 4° C. for 5 min. The ethanolic extracts will be pooled, evaporated to dryness and stored at −20° C. cGMP will be measured by scintillation proximity immunoassay (Amersham). All experiments will be performed in triplicate.

Compounds that pass the test in hippocampal cultures will also be tested in adult mice, following an assessment of acute toxicity to determine the dose of compound to be administered to the animal (see “toxicity tests” below). Animals will be treated with the PDE inhibitor, samples will be collected, homogenized immediately, and sonicated in the BIOTRAK cGMP enzyme immunoassay kit buffer containing 4 mM EDTA (Amersham, Ill.); samples will be centrifuged (12,000×g, for 5 min) to measure cGMP in the supernatant using the kit (results will normalized to the pellet protein levels with Lowry's procedure). If an increase in cGMP levels is found following PDE inhibitors and the inhibitor or its metabolites is detected in the dialysate, the candidate compound will be deemed as active.

Computational Strategy

General studies. To discover new drug candidates that bind to PDE5, medicinal chemistry efforts will be aided by computational modeling. A rational design approach, such as a structure-based virtual screening described in Xiong et al., (“Dynamic structures of phosphodiesterase-5 active site by combined molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical calculations,” Dec. 27 2007, J Comp Chem [Epub ahead of print]), can help to maximize the chances of finding new drugs that associate with amino acid residues F787, L804, I813, M816, or a combination thereof of PDE5 (Card et al., 2004, Structure, 12:2233-47). The computation protocol can be used for preliminary docking studies using the structures represented by the 4 major classes of scaffolds shown on FIG. 13. The computational results can qualitatively show whether some of the structures or their structural variants fit the binding site of PDE5.

General ADMET Considerations

Optimization with respect to ADMET properties of the library members should be considered at the early stage of the drug discovery to guide synthetic efforts [167, 168]. For design purposes, med chem filters (MCF) will be used [169, 170](e.g. presence of reactive, unstable, or toxicophore groups); compliance of the designed compounds will be controlled by the Lipinski “rule of five” [171, 172][it states: 1) five or fewer hydrogen bond donors; 2) ten or fewer hydrogen bond acceptors; 3) molecular weight less than 500; 4) calculated logP less than or equal to 5]; polar and lipophilic surface areas will be kept optimal for solubility and cell-permeability properties [173], and optimal bioavailability score [174] of potential inhibitors utilizing ADMET Predictor [149].

ADMET Predictor [149], an advanced ADMET structure-property prediction program that includes additional ADME predictive models beyond the well known rule of five, can be used to predict potential ligands via flexible docking. The program predicts all of the important properties critical to oral absorption (including pKa's), as well as several pharmacokinetic properties and many aspects of toxicity [149]. The BBB penetration will also be theoretically estimated by calculating the polar surface area (PSA) and the oil/water partition coefficient (logP) of each candidate compound and using the well-established quantitative structure-activity relationship (QSAR) and artificial neural network (ANN) models [150, 151]. These QSAR and ANN models have demonstrated that the BBB penetration of a compound is determined by the PSA and logP (or PSA and molecular weight) of the compound, both of which can be calculated conveniently by ADMET Predictor. Usually, compounds that can cross the BBB should have a molecular mass less than 450 Da and a PSA smaller than 90 Å^(2 [)150].

Such considerations are relevant to ensuring the ability of compounds to penetrate the membrane thus allowing for studies at both the cellular and animal levels. It should be noted, though, that molecules computationally predicted to be drug-like are not “automatically drug-like” [175] as there are too many different mechanisms and parameters that affect the actual in vivo activity. To allow for further modifications that can be required to improve ADMET profile, a major emphasis on using “lead-likeness” [176, 177] criteria will be placed. The activity and ADMET profiles of the resulting “lead-like” compounds can be later improved via additional rounds of computer modeling and medicinal chemistry efforts, and a variety of cell-based assays for cellular and molecular pharmacology and in vitro and in vivo toxicology.

Example 3 Identification of PDE5 Inhibitors which are Optimized for AD—Determination of Whether Compounds have Good P K, Bioavailability, and Brain Penetration

Pharmacokinetic assays that need to be performed when developing CNS drugs will include the measurement of a) bioavailability and b) brain uptake. They will be carried out in mice that will be i.p. injected with the candidate compounds (for final drug candidates PK tests will be also performed using p.o. and i.v. routes of administration). 5-6 mice/sex will be used for each time-point. For the assessment of bioavailability (concentration of compound in the blood as a function of time after administration), blood samples will be obtained from test animals following a single acute administration. The time course study after drug administration will include at least six points (5 minutes, 15 minutes, 1 hour, 2 hours, 5 hours, and 24 hours). The animals will be anesthetized with pentobarbital (50 mg/kg). Blood will be harvested by intracardiac puncture, collected in heparanized tubes, and plasma obtained by centrifugation. Samples will be analyzed by LC-MS to measure the amounts of the candidate compound and metabolites. An indication of brain uptake and blood brain barrier penetration will be obtained by tissue extraction of the candidate compound from brain following perfusion with PBS of the mice. Briefly, brain homogenates will be centrifuged 12,000×g for 10 min. The compound will be isolated by solid phase extraction, then analyzed by HPLC and measured using LC-MS. Pattern of time dependent changes in brain concentration will be compared with that of blood concentration. Similar patterns will be indicative of the fact that brain uptake reflects concentration of the blood. A peak brain/blood concentration ratio>1 will show that brain uptake for the compound is comparable with that of known CNS drugs in clinical use. For example, the brain/blood ratio for minaprine, a 6-phenylaminopyridazine CNS drug, is >2 [178].

General considerations: Of note, sildenafil has been shown to cross the BBB [179], and the efficacy of sildenafil on the AD animal models further demonstrates that PDE5 inhibitors can achieve brain penetration for a CNS target. While there is no “golden rule” for brain penetration, empiric correlations show the importance of a molecular mass under 400-500 Da, 8 or fewer hydrogen bonds, and the presence of basic amines rather than carboxylic acids. Also, a variety of methods including computational approaches (see “Stage #2” of “Computational design” above) have been developed to assess CNS penetration of drug candidates. Also, PDE5 is an intracellular enzyme; PDE5 inhibitors must cross the cell membrane to increase cGMP and thus cell based screening for PDE5 activity will also address the issue of absorbance. Finally, the structure of the candidate drug can support a chemistry-based approach to BBB penetration. For example, the polar functional groups on a water-soluble, non-CNS penetrating drug can be masked by introducing lipid-soluble moieties, or the water-soluble drug can be conjugated to a lipid-soluble drug carrier. Ideally, the new drug or the prodrug is metabolized within the brain and converted to the parent drug. This chemistry-based approach has been used successfully in solving the BBB drug-delivery problem in clinical practice [180]. In conjunction with the computational methods described at stage #2 it should provide reliable prediction of BBB access.

Ascertaining Whether Newly Identified Compounds are Safe

Before determining drug efficacy in the APP/PS1 mouse, but after synthesis of sufficient quantities of compound and a determination of a formulation for delivery, data addressing the rudimentary pharmacokinetic properties and toxicity of the compound will be generated. It has been estimated that over half of all drugs fail to reach the market because of problems with ADMET [181]. Therefore, before embarking on a course of costly animal toxicology, recent advances in in vitro ADMET testing will be utilized to screen compounds with a quick, inexpensive battery of assays performed by Charles River Laboratories. Two areas will be focused on that have resulted in the withdrawal of many drugs from the market and that can sometimes affect an entire chemical series: drug-drug interactions (liver metabolism), hERG channel blockage (cardiac dysfunction). To test for drug-drug interactions related to hepatotoxicity (the leading cause of drug withdrawal during the past 25 years and especially important for a heavily-medicated Alzheimer's population) [182], the Cytochrome P450 inhibition assay will be used. Cytochrome P450 is an important component of liver metabolism. Moreover, there are pharmacological interactions between PDE5 inhibitors and other medications metabolized by the cytochrome P450 (P3A4 isoform), such as the azole antifungals, erythromycin and the HIV protease inhibitors. Thus, the IC₅₀ data gathered from this ELISA assay will allow for the elimination of compounds that inhibit isozymes of CYP450. To test for hERG channel blockage, which impairs proper cardiac electrophysiology and can lead to Torsades-de-pointes and fatality, the rubidium flux method will be used to assess whether lead compounds affect ion flow through these important cardiac channels.

Next, acute toxicity will be evaluated. All clinical signs, time of onset, duration, reversibility of toxicity and mortalities will be recorded. Animals will be observed periodically during the first 24 hrs with continuous monitoring given to the first 4 hrs, then at least once a day for 14 days or until they die to check food and liquid intake, weight, as well as locomotion and exploratory behavior.

Maximum tolerated dose (MTD) and chronic toxicity will also be evaluated. MTD will be computed as the maximum administered dose that does not produce any toxicity effect in terms of malaise or death (body weight will be monitored over time). Chronic toxicity will assessed at the MTD. All clinical signs, time of onset, duration, reversibility of toxicity and mortalities will be recorded.

The occurrence of chronic toxicity signs will be assessed for at least 1 month after the end of the treatment. Gross necropsies will be performed in all animals, including those sacrificed moribund, found dead, or terminated at 14 days after the acute treatment or at the end of the chronic treatment over 30 days. Gross evaluation at necropsy will include weights and measurements of organs. The color of the organs will be noted to determine if there is fatty change, hemorrhage, pigment deposition or other changes. Organs will be palpated and directly visualized to examine for lesions and changes in consistency such as abnormal growths, fibrosis, necrosis, or fat deposition. Histopathologic evaluation of liver, kidney, brain and muscle will be performed.

Liver sections will be evaluated for portal and hepatocellular inflammation, bile ductular proliferation, hepatocellular injury including apoptosis and necrosis, fibrosis, steatosis, hypertrophy, pigment deposition (bile, iron, and copper) and oncocytic (mitochondrial proliferative) changes. Slides will be stained with hematoxylin and eosin and trichrome for initial review.

Renal sections will be examined with hematoxylin and eosin, trichrome and periodic acid Schiff (PAS). Glomeruli, vessels, tubules, collecting ducts and interstitium will be evaluated for cellularity, inflammation, collagen deposition/fibrosis/sclerosis. It will be determined if there is proximal tubular epithelial cell damage or renal papillary necrosis, some of the more common nephrotoxic effects.

Neurotoxicity will be evaluated in all regions of the brain, including neocortex, striatum, thalamus, hippocampus, cerebellum, brain stem and spinal cord. The brain will be examined for cytoarchitecture, neuronal loss (apoptosis and necrosis), inflammation, axonal degeneration, gliosis, and myelination. Hematoxylin and eosin stained slides will be used for general assessment and additional stains if needed will include Luxol fast blue-PAS for myelination, GFAP (glial fibrillary acidic protein) for astrocytic response, and CD68 for microglial response.

Muscle will be evaluated with hematoxylin and eosin to examine for neurogenic or myopathic atrophy, necrotic fibers, regenerative fibers, fat deposition, or inflammation. Trichrome stain will be used to determine fibrosis. If changes are determined during the initial screening, frozen sections will be made to evaluate fiber type distribution with ATPase stains. In vitro cytotoxicity assays will be carried out to evaluate cell viability after administration of the drug in primary neuronal cultures using fluorescein diacetate method. Motor, sensor, motivational and cognitive performances will be monitored during both acute and chronic toxicity evaluations using the visible and hidden platform testing, as well as gross behavioral evaluation (exploratory behavior, PICA, feeding, distress). To avoid causing excessive pain or tissue damage in the animal, pharmaceuticals with irritants or corrosive character will not be administered in concentration that can produce severe toxicity solely from local effects.

General considerations: Although in vivo toxicity is a very difficult property to predict, some general strategies will be followed. Functionality that can render a molecule electrophilic (i.e. alkyl halides or Michael acceptors) would be addressed immediately. Such functionality is commonly found to result in toxicity. For example, if a hit contained a bromomethyl group, derivatives would be prepared that eliminate the bromide, replacing it with an electron-withdrawing and/or hydrophobic group (but not a good leaving group), such as trifluoromethyl, or eliminating the methylene between the bromine and the other substituent attached to the methylene (i.e. transforming a benzyl bromide to a bromophenyl).

Metabolic stability is also difficult to predict a priori. However, functionality that is commonly known to be metabolically unstable (i.e. esters) would be replaced with known bioisosteres [183]. Another common pathway leading to lower metabolic stability is aromatic ring oxidation. Therefore, during the SAR studies aromatic and heterocyclic rings will be rendered less electron-rich by the strategic placement of electron-withdrawing groups (e.g. F or Cl) or by substitution (i.e. replacing a phenyl ring with a pyridine ring). In cases where this change does not result in decreased efficacy, a significant increase in metabolic stability can be realized. In addition, efforts would be made to eliminate functionality known to produce metabolites that are prone to bioconjugation [184]. Such metabolites can be hepatotoxic and limit the usefulness of the compound which will be discarded.

Compound analysis. All compounds synthesized and tested for biological activity will be fully characterized and purified to >95% as determined by HPLC and ¹H NMR. Furthermore, additional analytical techniques (i.e. ¹³C NMR, IR, melting point, MS and/or elemental analysis) will be used to determine structure and purify. In the case of optically pure materials, the purity will be assessed by chiral stationary-phase HPLC. In certain cases where structural uncertainty remains other techniques (i.e. 2-D NMR, and x-ray crystallography) will be utilized.

Example 4 Screening of the New PDE5 Inhibitors by Selecting Compounds that Rescue Synaptic Dysfunction in APP/PS1 Mice

Synaptic dysfunction is a major hallmark of AD [1]. Several animal models of AD have become available during the last 12 years. Since even in the fastest model, AD pathology does not start before the end of the 2^(nd) month, it has been necessary to wait at least until this age to inject drugs into the animal to assess whether they are beneficial to synaptic impairment, plaque formation and increase of Aβ levels. Such in vivo approaches can be labor intensive. An alternative approach is achieved by the use of cell cultures from Tg animals which provide a new, fast, efficient and reproducible in vitro method for the screening and testing of compounds for the treatment and therapy of AD or Aβ-associated diseases (see U.S. patent Ser. No. 10/980,922). These candidates will be examined to see whether they can rescue changes in basal number of active boutons and glutamate-induced long-lasting increase in active bouton number in APP/PS1 cultures. This method is relatively fast and easy to perform [93, 185].

The PDE5 inhibitors will be examined if they re-establish normal numbers of active boutons and glutamate-induced increases in active boutons in cultures from APP/PS1 mice. Next, these results will be validated in hippocampal slices to see if the enhancers re-establish normal LTP in the CA1 region of slices from APP/PS1 mice.

Based on a med/chem analysis of existing PDE5 inhibitors, four classes of scaffolds have been identified that can serve for the development of new PDE5 inhibitor candidates. These compounds are being screened and optimized using the computational models described herein. Thus, new PDE5 inhibitors will be identified with a) high specificity and potency, b) great CNS penetration, and c) safety. The following fundamental 3 endpoints will be focused on: a) identification of compounds with high affinity for PDE5 and good selectivity relative to other PDEs; b) determination of whether such compounds have good PK, bioavailability and brain penetration; c) ascertaining whether compounds that meet the aforementioned criteria are safe.

Experimental design: Based on the finding that APP/PS1 cultures show an increase in the basal number of functional presynaptic release sites (see FIG. 22B), the compounds will be screened as shown by MedChem studies to select those that can re-establish normal basal number of active boutons. The number of active boutons, with and without PDE5 inhibitor treatment, will be examined in cultures from double Tg- and WT-littermates. 10-day old cultures from APP/PS1 and WT littermates will be treated for 4 days to test whether the compounds can rescue the increase in active bouton number. 10-day old cultures from APP/PS1 and WT littermates will also be treated for 20 min to test whether a short treatment rescues the increase in functional active boutons. If there is no difference in basal active bouton number between compound-treated cultures from double Tg and WT animals, but cultures from double Tg mice treated with vehicle alone show increased basal active bouton number, compounds will be deemed blockers of the development of changes in basal number of functional boutons in cultures from AD animal models.

Lack of a glutamate-induced increase in the number of active boutons is another phenomenon that occurs in cultures from APP/PS1 mice (see FIG. 22C). The new compounds will be examined as to whether they can rescue the impairment of this plastic change. The same strategies will be used as for the basal number of active boutons. Briefly, cultures from APP/PS1 and WT littermates will be treated for 4 days from day 10, or for 20 min on day 10 before evoking the glutamate-induced increase in active bouton number. Re-establishment of the glutamate-induced increase in active bouton number will be examined. If this is observed, the compounds will be deemed to being able to rescue impairment of synaptic plasticity in cultures from APP/PS1 mice.

Methods

Double Tg mice will be obtained by crossing APP(K670M:N671L) with PS1(M146L) (line 6.2) animals. The genotype will be identified by PCR on tail samples [186-188]. Primary cultures will be prepared from one-day-old mouse pups as previously described (see Ninan et al [189]).

Vesicle Cycling studies will be done 7-21 days after plating (see detailed description in Ninan et al [189]). Briefly, loading of FM 1-43 will be induced by changing the perfusion medium from normal bath solution to hyperkalemic solution with 5 μM FM 1-43 for 45 sec. ADVASEP-7 (1 mM) will be introduced for 60 sec in the washing solution at 1 and 6 min of washing. Unloading will be performed with multiple 15 sec applications of hyperkalemic solution (without FM 1-43). The difference between the images before and after multiple exposures to hyperkalemic solution will give the measure of FM 1-43 stained vesicles [see FIG. 22A]. The number of active boutons per uniform length of randomly selected neurites (15×6.85 μm field) at 12 μM from the cell body will be measured in blind. Plasticity will be induced through glutamate (200 μM in Mg²⁺ free bath solution for 30 sec). Staining and destaining procedures will be repeated 30 min after glutamate. All images will be acquired using Nikon D-Eclipse C1 confocal microscope. Total number of boutons from randomly selected fields (30.8×30.8 μm) will be blindly assessed using NIH Image (v. 1.61).

Electrophysiological Analysis will be performed on males (see detailed description in Gong et al, [83]). 400 μm slices will be cut with a tissue chopper and maintained in an interface chamber at 29° C. for 90 min prior to recording. Briefly, CA1 fEPSPs will be recorded by placing both the stimulating and the recording electrodes in CA1 stratum radiatum. BST will be assayed either by plotting the stimulus voltages against slopes of fEPSP, or by plotting the peak amplitude of the fiber volley against the slope of the fEPSP. For LTP experiments, a 15 min baseline will be recorded every min at an intensity that evokes a response ˜35% of the maximum evoked response. LTP will be induced using O-burst stimulation (4 pulses at 100 Hz, with the bursts repeated at 5 Hz and each tetanus including 3 ten-burst trains separated by 15 sec).

Statistical Analyses will be carried out as described herein.

Example 5 Screening of PDE5 Inhibitors Selected Through Tests on Synaptic Function to Examine Whether they Prevent Cognitive Abnormalities in APP/PS1 Mice

Enhancers of the NO/sGC/cGMP/PKG/CREB pathway can rescue the cognitive deficits observed in APP/PS1 mice of 3 and 6 months of age. New PDE5 inhibitors screened in Tg cultures and slices will be determined as to whether they can protect APP/PS1 mice against impairments of spatial working memory, reference memory and contextual fear learning. Treatment with the new PDE5 inhibitors will be examined to see if they have beneficial effects on abnormal cognition in APP/PS1 mice.

Experimental design: In a first series of experiments, spatial working memory, a type of short term memory that is impaired at early stages in patients affected by AD and APP/PS1 mice, will be tested using the RAWM. Next, associative memory, a type of contextual memory that is tested with FC and is impaired at the age of 3 month in APP/PS1 mice, will be examined. Finally, reference memory, a type of long-term memory that is tested with the MWM and is impaired in APP/PS1 mice at the age of 6 months, will be assessed. In addition, controls will be conducted with the visible platform task, sensory threshold and cued conditioning tests. The treatment will be performed with the same timing as in the preliminary studies (i.e. immediately after training). Conditions to be tested include: APP/PS1 and WT treated with PDE5 inhibitors, APP/PS1 and WT treated with vehicle. After behavioral testing mice will be sacrificed and their blood and brains used for measurement of Aβ levels. As a control for effectiveness of PDE5 inhibition, hippocampal cGMP levels in APP/PS1 mice will be measured after administration of the compounds. If the compounds have a beneficial effect, there should be no difference or little difference in the RAWM, and/or MWM, and/or FC tasks between compound-treated Tgs and WT littermates, whereas vehicle-treated double Tgs should show abnormal L&M. Compound-treated WT mice should show normal learning. No difference is expected in speed and latency to the platform (visible platform test), as well as in the cued conditioning for the various groups of mice. No difference is also expected in sensory perception of the shock for the various groups of mice. These results will indicate that treatment with these compounds can prevent the development of cognitive abnormalities in AD animal models. There is also the possibility that the compounds can ameliorate one type of memory and not the other. The beneficial effect of the compounds is limited to that type of memory.

In these studies, the beneficial effect of sildenafil on RAWM and FC has been observed to last beyond the duration of the application of the drug. To address whether this prolonged effect on RAWM and FC is observed using the newly identified PDE5 inhibitors, one group of 3-month-old APP/PS1 mice will be used that will be divided into two subgroups that will be treated with the compounds and vehicle, respectively. The treatment will last for 3 weeks. WT littermates will serve as controls and receive the same treatment. As the animals will be 6 months old, they will perform the full battery of behavioral tests, including RAWM, MWM and contextual FC, as well as visible platform testing, sensory threshold testing and cued conditioning. Then the animals will be sacrificed for measurement of hippocampal cGMP levels, and blood and brain Aβ levels. If the compounds will re-establish normal cognition, the beneficial effect of these compounds will be deemed to act on the cognitive impairment of adult AD animal models that lasts beyond the drug application.

Methods

Animals to be used in these studies have been described herein.

Behavioral studies: Experiments will be performed in blind only on male animals to reduce variability.

Spatial working memory. This type of short-term memory can be studied with the RAWM test. The task has proven informative in the analysis of other Tg models of AD [23, 33, 76, 211]. Briefly, the RAWM will consist of a tank filled with opaque water by powdered milk. Walls will positioned so as to produce six arms, radiating from a central area. Spatial cues will be present on the walls of the testing room. At the end of one of the arms will be a clear 10 cm submerged platform that will remain in the same location for every trial on a given day, but will be moved about randomly from day to day. For each trial the mouse will start the task from a different randomly chosen arm. The mouse cannot use its long-term memory of the location of the platform on previous days, but must rely on the short-term memory of its location on the day in question based on spatial cues that are present in the room. Each trial will last 1 min and errors will be counted each time the mouse will enter the wrong arm or will need more than 10 sec to reach the platform. After each error the mouse will be pulled back to the start arm for that trial. After 4 consecutive acquisition trials, the mouse will be placed in its home cage for 30 min, then returned to the maze and administered a 5^(th) retention trial. Testing will be considered completed when the WT mice make the same number of errors during the 4^(th) and 5^(th) trial. The scores for each mouse on the last 3 days of testing will be averaged and used for statistical analysis. Visible-platform training to test visual and motor deficits will be performed in the same pool but without arms, with the platform marked with a black flag and positioned randomly from trial to trial. Each animal will be allowed to swim for 1 min. Time to reach the platform and speed will be recorded.

Reference memory. This long-lasting type of memory will be tested with the MWM, as previously described [23]. Briefly, the test will be performed in the same pool as above but without arms. The pool will be divided into 4 sections. The mouse will start from one section and will have to find a hidden platform beneath the surface of the water. The location of the platform will remain constant throughout the different days. Time required to reach the hidden platform (latency) will be recorded. The training will be followed by 4 probe trials with the platform moved to test the retention of the spatial memory. The percent of time spent in the quadrant that used to contain the platform will be recorded and analyzed with a video-tracking system (HVS Image, UK).

FC. This associative memory test is much faster than other behavioral tasks that require multiple days of training and testing [33]. The conditioning chamber will be in a sound-attenuating box. The conditioning chamber will have a 36-bar insulated shock grid removable floor. For the cued and contextual conditioning experiments, mice will be placed in the conditioning chamber for 2 min before the onset of a discrete tone (CS) (a sound that lasted 30 s at 2800 Hz and 85 dB). In the last 2 s of the CS, mice will be given a foot shock (US) of 0.50 mA for through the bars of the floor. After the CS/US pairing, the mice will be left in the conditioning chamber for another 30 s and will then be placed back in their home cages. Freezing behavior, defined as the absence of all movement except for that necessitated by breathing, will be scored using the Freezeview software (MED Ass. Inc.). To evaluate contextual fear learning, freezing will be measured for 5 min (consecutive) in the chamber in which the mice will be trained 24 hr after training. To evaluate cued fear learning, following contextual testing, the mice will be placed in a new context (triangular cage with smooth flat floor and with vanilla odorant) for 2 min (pre-CS test), after which they will be exposed to the CS for 3 min (CS test), and freezing will be measured. Sensory perception of the shock will be determined through threshold assessment, as described [33].

Determination of cGMP and cAMP: The method has been described in the “Evaluation of drug activity” “tests in adult mice” section (for cAMP, BIOTRAK cAMP enzyme immunoassay kit buffer will be used).

Determination of Aβ levels will be performed on homogenates of frozen hemi-brains as previously described [23]. Homogenate will be mixed with formic acid, sonicated, and spun at 50,000 rpm at 4° C. Supernatant will be diluted in neutralization solution. The neutralized material will be stored at −80° C. before loading onto ELISA plates. Aβ will be trapped with either monoclonal antibody to Aβ40 (JRF/cAβ40/10) or Aβ42 (JRF/cAβ42/26) and then detected with horseradish peroxidase-conjugated JRF/Aβtot/17 [212]. ELISA signals will be reported as the mean±s.e.m. of two replica wells in fmol amyloid-β per mg protein (determined with the BCA Protein Assay Reagent Kit, PIERCE), based on standard curves using synthetic Aβ40 and Aβ42 peptide standards (American Peptide).

Blood will be harvested in a tube containing 10 mM EDTA, centrifuged at 4000 rpm for 5 min at 4° C. Plasma will be stored at −80° C. before loading onto ELISA plates.

Statistical analyses: For all experiments mice will be coded to blind investigators with respect to genotype and treatment. Results will be expressed as Standard Error Mean (SEM). Level of significance will be set for p<0.05. Results will be analyzed with ANOVA with post-hoc correction with drug or genotype as main effect. Experiments will be designed in a balanced fashion, and mice will be trained and tested at each of the different conditions in 3 or 4 separate experiments. For probe trials, data will be analyzed with ANOVA for repeated measures for percent of time spent in the quadrant where the platform is located during training with respect to other quadrants, followed by planned comparisons to confirm if mice spend more time in the target than in adjacent quadrant to the right, left, or opposite from the target quadrant.

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Example 6 Quinoline Compounds and Pharmacology Studies

Electrophysiological Protocol

Following cutting hippocampal slices were transferred to a recording chamber where they were maintained at 29° C. and perfused with artificial cerebrospinal fluid (ACSF) continuously bubbled with 95% O₂ and 5% CO₂. The ACSF composition in mM was: 124.0 NaCl, 4.4 KCl, 1.0 Na₂HPO₄, 25.0 NaHCO₃, 2.0 CaCl₂, 2.0 MgSO₄, 10.0 glucose. CA1 fEPSPs were recorded by placing both the stimulating and the recording electrodes in CA1 stratum radiatum. BST was assayed either by plotting the stimulus voltages against slopes of fEPSP, or by plotting the peak amplitude of the fiber volley against the slope of the fEPSP. A 15 min baseline was recorded every min at an intensity that evokes a response ˜35% of the maximum evoked response. LTP was induced using q-burst stimulation (4 pulses at 100 Hz, with the bursts repeated at 5 Hz and each tetanus including 3 ten-burst trains separated by 15 sec). Responses were recorded for 1 hr after tetanization and measured as field-excitatory-post-synaptic potential (fEPSP) slope expressed as percentage of baseline.

In these experiments YF012403 (the cyclopropyl lead compound) was directly given to the hippocampal slices through the perfusion system for 10 min prior to the theta burst. Aβ42 was given for 20 minutes prior to the theta burst. Oligomeric Aβ42 was prepared as described previously (Stine et al., 2003). Briefly, the lyophilized peptide (American Peptide) was resuspended in 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; Sigma, St. Louis, Mo.) to 1 mM. The solution was aliquoted, and the HFIP was allowed to evaporate in the fume hood. The resulting clear peptide film was dried under vacuum in a SpeedVac and stored at −20° C. Twenty-four hours before use, the aliquots were added to dimethylsulfoxide (DMSO; Sigma) and sonicated for 10 min. Oligomeric Aβ-42 was obtained by diluting Aβ-42-DMSO into ACSF concentration, vortexed for 30 s, and incubated at 4° C. for 24 h. Before use, this compound was added to ACSF to obtain 200 nM.

Acute Toxicity Profile

Timeline: 24 h-7 days. In the timeline, no fatal effects were observed.

Dosage:

-   -   Single dose at 500 mg/kg, i.p.     -   Single dose at 1000 mg/kg, i.p.     -   Single dose at 2000 mg/kg, p.o.

Species: mouse

Compounds

A new class of quinoline-containing compounds have been synthesized which have excellent PDE5 inhibitory potency, high selectivity, reasonable pharmacokinetics and good permeability to the blood-brain-barrier (BBB). These compounds may be used to minimize the side effects for AD patients, the third most costly disease in the U.S. The compounds of the invention may also be used to treat erectile dysfunction (ED), pulmonary hypertension, cardiovascular disorder, diabetes, and GI disorders.

The leading compounds are an 8-cyclopropyl quinoline derivative (YF012403) and an 8-dimethylaminoquinoline (YF016203) derivative. The IC₅₀ of these compounds to PDE5 are 1.2 nM and 4.5 nM, respectively. For example, in BABL/c mice, the YF012403 compound half-life is 1.04 h in the brain and 1.33 hr in the plasma as compared to Sildenafil (a known PDE5 inhibitor) which has a brain half-life of 0.84 h and a plasma half-life of 1.21 h. Distribution of YF012403 in brain tissue versus that in the plasma (non-protein-bound free form; C_(Brain)/C_(Plasma)) is 0.41, which indicates that the penetration of the compound to the BBB is acceptable with respect to druggability. Thus, the compounds are potential candidates for treatment of AD patients.

YF012403 has high potency (IC₅₀=0.27 nM), and excellent selectivity for PDE5 over other PDE isoforms (FIG. 27). In addition, it penetrates the BBB after p.o. administration with a T_(max)=0.5 hr and a C_(max)=385 ng/g at a dosage of 50 mg/kg. Furthermore, the compound is safe up to 2 g/kg (p.o.) in acute toxicity test. Most importantly, it shows both ex vivo and in vivo efficacy: it ameliorates LTP in hippocampal slices treated with Aβ42 and contextual fear memory in mice infused with Aβ42. YF012403 is biologically active in tests of synaptic and cognitive function following Aβ elevation.

Using YF012403 as a lead candidate, we will design and synthesize PDE5 inhibitors bearing different moieties at the C3 and the C8 positions, as well as other parts of the quinoline. It is noted that N, and S groups substituted at the C8 position of the quinoline (see also FIG. 50), have not been previously reported.

General Synthesis Method of Scheme A

The incentive compounds of formulas XIII′ and XIV′ can be prepared conveniently according to the synthetic sequence as shown in Scheme A (FIG. 38).

As shown in FIG. 38, starting from commercial available 4-amino-3-bromobenzonitrile (I′), the key intermediate, substituted 4-hydroxyquinoline III′, is conveniently prepared by reaction of aniline I′ with diethyl ethoxymethylenemalonate, followed by an intra-molecular cyclization reaction at a high temperature. The substituted 4-hydroxyquinoline III′ is then allowed to react with arylalkyl halide, aroyl halide or arylsulfonyl halide to afford 8-bromoquinoline V′. Alternatively, by reaction with POCl₃, the 4-hydroxyquinoline is readily converted to the corresponding 4-chloroquinoline VI′, which reacts either with arylalkylamine to afford key intermediate VIII′, or with ammonia directly to yield 4-aminoquinoline IX′. The 4-aminoquinoline is then allowed to react with arylalkyl halide, aroyl halide or arylsulfonyl halide to give another key intermediate X′. Starting from the key intermediates V′, VIII′, X′, which are represented by formula XI′, the incentive formula XIII′ is prepared by coupling of cycloalkylboronic acid or substituted amine with the 8-bromoquinoline XI′, followed by reduction of the ethyl ester to provide the resulting intermediate XIII′. Through substitution by nucleophiles such as substituted amines, or reaction with electrophiles such as alkyl, acyl or sulfonyl halides, the incentive formula XIII′ is conveniently converted to incentive formula XIV′.

Synthesis Examples of Compounds

The following examples are offered for illustrative purpose for the incentive compounds and intermediates, and are not intended to limit the scope of the claims in any manner. Those skill of the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

Example 1

The synthesis of compound 9a within the invention is outlined in Scheme I, with the details of the individual steps given in FIG. 39.

Preparation of diethyl 2-((2-bromo-4-cyanophenylamino)methylene)malonate (Intermediate 3a)

To a solution of 5.00 g (25.4 mmol) 4-amino-3-bromobenzonitrile in 30 mL of toluene was added 8.23 g (38.1 mmol) of diethyl ethoxymethylenemalonate (2a). The mixture was then heated to reflux for overnight with a condenser open to the air. The resulting solution was cooled down to room temperature and poured into 100 mL of hexanes. The white precipitate was collected and washed with hexanes (30 mL×3) to yield 11.9 g of an off-while solid as the desired product. MS ESI (m/z) 367 (M+1)⁺.

Preparation of ethyl 8-bromo-6-cyano-4-hydroxyquinoline-3-carboxylate (Intermediate 4a)

100 mL of diphenyl ether was heated to reflux followed by addition of 5.00 g (13.6 mmol) diethyl 2-((2-bromo-4-cyanophenylamino)methylene)malonate in portions in 30 minutes. The resulting brown solution was reflux for another hour and then cooled down to room temperature. The precipitate was collected and washed with hexanes (15 mL×3) to give 5.69 g of a light brown solid as the desired product. MS ESI (m/z) 321 (M+1)⁺.

Preparation of ethyl 8-bromo-4-chloro-6-cyanoquinoline-3-carboxylate (Intermediate 5a)

The mixture of 3.85 g (12 mmol) and 50 mL of POCl₃ was heated to reflux for 48 hours. The solvent was removed in vacuum and co-distilled with CHCl₃ (50 mL) and toluene (50 mL×2). The resulting dark brown syrup was dissolved in 50 mL of CH₂Cl₂ and treated with Et₃N until pH>10. The dark-red solution was then allowed to go through a silica gel pad (3 cm×4 cm). The silica pad was washed with 100 mL of CH₂Cl₂. The filtrates were collected and concentrated to yield a brown solid, which was used in the next step directly without further purification.

Preparation of ethyl 8-bromo-4-chloro-6-cyanoquinoline-3-carboxylate (Intermediate 7a)

To the crude product of ethyl 8-bromo-4-chloro-6-cyanoquinoline-3-carboxylate obtained above was added 3.12 g (15 mmol) of (3-chloro-4-methoxyphenyl)methanamine hydrochloride (6a), 7.74 g of diisopropylethylamine and 50 mL of n-propanol. The resulting mixture was refluxed for 2.5 hours and then poured to 100 mL of ice-water. The precipitate was collected by filtration and washed by H₂O (30 mL x2) and ethanol (30 mL×3) to give 5.0 g of a yellow solid as the title compound. MS ESI (m/z) 474 (M+1)⁺.

Preparation of ethyl 4-(3-chloro-4-methoxybenzylamino)-6-cyano-8-cyclopropy-quinoline-3-carboxylate (Intermediate 8a)

Under nitrogen, to the solution of 475 mg (1 mmol) of 8-bromo-4-chloro-6-cyano-quinoline-3-carboxylate in 5 mL of dry toluene was added 129 mg (1.5 mmol) of cyclopropylboronic acid, 58 mg (0.05 mmol) of (tetrakis(triphenylphosphine) palladium (0) and 815 mg (2.5 mmol) of Cs₂CO₃. After the mixture was refluxed overnight, the precipitate in the solution was removed by filtration. The filtrate was concentrated and purified by flash chromatography (ethyl acetate:hexanes=1:4) to yield a 366 mg of a yellow solid as the desired compound. MS ESI (m/z) 436 (M+1)⁺.

Preparation of 4-(3-chloro-4-methoxybenzylamino)-8-cyclopropyl-3-(hydroxymethyl)-quinoline-6-carbonitrile (Compound 9a)

Under nitrogen, to the solution of 180 mg (0.43 mmol) of intermediate 8 in 5 mL of dry THF was added 2.2 mL (2.2 mmol) of lithium tri(tert-butoxy)aluminum hydride (1 M in hexane). The resulting solution was refluxed overnight and then quenched with 1 mL of MeOH. 30 minutes later, the mixture was poured to a separatory funnel, followed by addition of 150 mL of CH₂Cl₂ and 50 mL of 1N NaOH. The organic layer was separated, washed with 1N NaOH (50 mL) and dried over MgSO₄. The solid was filtered off. Concentration of the filtrate gave 156 mg of a yellow solid as the incentive compound. ¹H NMR (DMSO-d₆, 300 MHz): δ 8.69 (d, J=1.2 Hz, 1H), 8.48 (s, 1H), 7.42 (t, J=7 Hz, 1H), 7.37 (d, J=2.1 Hz, 1H), 7.33 (d, J=1.2 Hz, 1H), 7.21 (dd, J₁=8.4 Hz, J₂=2.1 Hz, 1H), 7.08 (d, J=8.4 Hz, 1H), 5.38 (t, J=5.1 Hz, 1H), 4.79 (d, J=7 Hz, 2H), 4.43 (d, J=5.1 Hz, 2H), 3.79 (s, 3H), 3.09-3.14 (m, 1H), 1.02-1.08 (m, 2H), 0.72-0.87 (m, 2H); MS ESI (m/z) 394 (M+1)⁺.

Example 2

The synthesis of compound IIa within the invention is outlined in Scheme II, with the details of the individual steps given in FIG. 40.

Preparation of ethyl 4-(3-chloro-4-methoxybenzylamino)-6-cyano-8-(dimethylamino)-quinoline-3-carboxylate (Intermediate 10a)

Under nitrogen, to the solution of 475 mg (1 mmol) of 8-bromo-4-chloro-6-cyano-quinoline-3-carboxylate in 5 mL of dry toluene was added 11 mg (0.05 mmol) of palladium (II) acetate, 50 mg of (R)-BINAP, and 812 mg (2.5 mmol) of Cs₂CO₃, and 3 mL of the solution of dimethylamine in ethanol (5.6 M). After the mixture was refluxed overnight, the precipitate in the solution was removed by filtration. The filtrate was concentrated and purified by flash chromatography (ethyl acetate:hexanes=1:2) to yield a 140 mg of a yellow solid as the desired compound. MS ESI (m/z) 439 (M+1)⁺.

Preparation of 4-(3-chloro-4-methoxybenzylamino)-8-(dimethylamino)-3-(hydroxymethyl)-quinoline-6-carbonitrile (Compound IIa)

Compound IIa was prepared by a method analogous to that described in the preparation of compound 9a starting from ethyl 4-(3-chloro-4-methoxybenzylamino)-6-cyano-8-(dimethylamino)-quinoline-3-carboxylate. ¹H NMR (CDCl₃, 300 MHz): δ 8.37 (s, 1H), 7.90 (d, J=1 Hz, 1H), 7.31 (d, J=2 Hz, 1H), 7.16 (dd, J=8.7 Hz, J₂=2 Hz, 1H), 7.05 (d, J=1 Hz, 1H), 6.90 (d, J=8.7 Hz, 1H), 5.70 (t, J=4.2 Hz, 1H), 4.67 (s, 2H), 4.64 (d, J=4.2 Hz, 2H), 3.89 (s, 3H), 3.04 (s, 6H); MS ESI (m/z) 397 (M+1)⁺.

Intermediate 10a within the invention is also synthesized through the synthetic route outlined in Scheme III-A1, with the details of the individual steps given in FIG. 41.

Preparation of 3-(dimethylamino)-4-nitrobenzonitrile (Intermediate 13a)

The mixture of 16.7 g (100 mmol) of 3-fluoro-4-nitrobenzonitrile and 100 mL of the solution of dimethylamine in ethanol (5 M) was refluxed overnight. The resulting dark red solution was then poured in to 100 mL of ice-water. The precipitate was collected by filtration and washed by H₂O (50 mL×2) and ethanol (50 mL×2) to give 16.9 g of a organe needle crystal as the desired product. MS ESI (m/z) 192 (M+1)⁺.

Preparation of 4-amino-3-(dimethylamino)benzonitrile (Intermediate 14a)

To 16 g (84 mmol) of 3-(dimethylamino)-4-nitrobenzonitrile was added 1 g of palladium on carbon (10%, w/w) and 100 mL of methanol. The mixture was saturated with hydrogen and stirred at room temperature overnight. The palladium on carbon was then filtered off. Concentration of the filtration gave 12.8 of a dark-red solid as the desired product. MS ESI (m/z) 162 (M+1)⁺.

Preparation of diethyl 2-((4-cyano-2-(dimethylamino)phenylamino)methylene)malonate (Intermediate 15a)

Intermediate 15a was prepared by a method analogous to that described in the preparation of intermediate 3a starting from 4-amino-3-(dimethylamino)benzonitrile. MS ESI (m/z) 332 (M+1)⁺.

Preparation of ethyl 6-cyano-8-(dimethylamino)-4-hydroxyquinoline-3-carboxylate (Intermediate 16a)

Intermediate 16a was prepared by a method analogous to that described in the preparation of intermediate 4a starting from diethyl 2-((4-cyano-2-(dimethylamino)phenylamino)-methylene)malonate. MS ESI (m/z) 286 (M+1)⁺.

Preparation of ethyl 4-chloro-6-cyano-8-(dimethylamino)quinoline-3-carboxylate (Intermediate 17a)

Intermediate 17a was prepared by a method analogous to that described in the preparation of intermediate 5a starting from ethyl 6-cyano-8-(dimethylamino)-4-hydroxy-quinoline-3-carboxylate. MS ESI (m/z) 304 (M+1)⁺.

Preparation of ethyl 4-(3-chloro-4-methoxybenzylamino)-6-cyano-8-(dimethylamino)-quinoline-3-carboxylate (Intermediate 10a)

Intermediate 10a was prepared by a method analogous to that described in the preparation of intermediate 7a starting from ethyl 6-cyano-8-(dimethylamino)-4-hydroxy-quinoline-3-carboxylate. MS ESI (m/z) 439 (M+1)⁺.

Protocol of PDE Assay for Compound Screening

Materials: IMAPTM TR-FRET Screening Express with Progressive Binding Kit from Molecular Devices (R8160); FAM-Cyclic-3′,5′-GMP from Molecular Devices (R7507); PDE5 inhibitors.

Methods

Step 1: A dilution series of the inhibitors ranging from 300 μM to 10 μM in 1×PDE Assay Buffer are made. Subsequently, FAM-cGMP is diluted to 200 nM in 1×PDE Assay Buffer. PDE5A1 enzymes are then diluted to 0.125 ng/μl in 1×PDE Assay Buffer.

Step 2: The following components are added to a low binding black plate: a) 25 μl of 200 nM FAM-cGMP (Final concentration will be 100 nM); b) 5 μl of the compounds (Final concentration=30 pM to 1 μM); c) 20 μl of PDE5A1 (0.125 ng/μl) (Final amount=2.5 ng/reaction). The components are mixed and incubated at room temp. for 1 hour.

Step 3: A 1× reagent-binding buffer (75% 1× Binding Buffer A and 25% 1× Binding Buffer B) is then prepared followed by a Binding Solution that is prepared by diluting Binding Reagent with 1× reagent-binding Buffer (1:600). 120 μl of Binding Solution is then added to each well and the plate is incubated at room temperature for 1 hour.

Step 4: Fluorescence polarization is measured at excitation of 485 nm and emission of 520 nm in BioTek Synergy™ 2 microplate reader.

Protocol of Pharmacokinetics Testing

The pharmacokinetic studies were conducted in male BABL/c mice. The blood and brain samples were collected at predetermined times from three mice per time point. Six time points were measured for each compound: 0, 0.25, 0.5, 1.0, 2.0, and 4.0 hour. An LC-MS/MS method was developed to determine these compounds in plasma and brain samples.

Quantification was achieved by the internal standard method using peak area ratios of the analysis to the internal standard in plasma and brain. Concentrations were calculated using a weighted least-squares linear regression (W=1/x2). The major pharmacokinetic parameters were calculated and the brain-to-plasma distribution ratios were estimated.

Dose preparation and dose administration: The PDE5 inhibitor was prepared by dissolving the test article in 0.5% methyl cellulose to yield final concentrations at 5 mg/mL for PO administration. Sildenafil was prepared by dissolving the article in 0.2 M hydrochloric acid solution (pH=1) to yield final concentrations at 5 mg/mL. Dose volume for each test animal was determined based on the most recent body weight.

Sample Collection

Blood. Blood (approximately 250 μL) was collected via retro-orbital puncture into tubes containing sodium heparin anticoagulant at pre-dose (0 h) and 0.25, 0.5, 1.0, 2.0, and 4.0 hour from three mice per time point after administration. Mice were sacrificed by cervical dislocation after blood harvest. The plasma were separated via centrifugation (4° C., 3500 rpm, 10 min) and stored in −80° C. before analysis.

Preparation of plasma samples. Frozen unknown plasma samples were thawed at room temperature and vortexed thoroughly. With a pipette, 25 μL of plasma was transferred into a 1.5 mL Eppendorf tube. To each sample, 25 μL of methanol and 25 μL of the internal standard were added, followed by the addition of 100-μL methanol. The sample mixture was vortexed for approximately 1 min. After centrifugation at 11000 g for 5 min, the upper organic layer was transferred to a glass tube and evaporated at 40° C. under a gentle stream of nitrogen. Residues were dissolved in 150 L of the mobile phase, and mixed in a vortex mixer. A 20-μL aliquot of the resulting solution was injected onto the LC/MS/MS system for analysis.

Brain. Brains were collected immediately after mice death. The brains were excised, weighed, and rinsed by cold saline and then frozen at −80° C. until further process for LC/MS/MS analysis.

Preparation of brain samples. On the day of the assay, the frozen tissue samples were thawed unassisted at room temperature. When completely thawed, each tissue sample of 200 mg was weighed and placed into a plastic tube. Methanol (1.0 mL) was added to facilitate homogenization, which was conducted using a Fluko F6/10 superfine homogenizer for approximately 1 min. Then, the homogenized samples were vortexed for 1 min. A 25-μL aliquot of the homogenized samples was transferred into an Eppendorf tube. To each sample, 25 μL of methanol and 25 μL of the internal standard were added. The sample mixture centrifuged at 11000 g for 5 min. A 20-μL aliquot of the supernatants was diluted to 80 μL or 60 μL with the mobile phase and a 10-μL aliquot was injected onto the LC/MS/MS system for analysis.

Example 7 Quinoline Derivatives can be Developed as Potent and Selective PDE5 Inhibitors for the Treatment of AD

Our findings support that inhibition of PDE5 can be beneficial against cognitive loss in AD. However, none of the existing commercially available inhibitors, including sildenafil, are optimized for the CNS. Moreover, even the non-commercially available synthesized inhibitors have not been fully characterized for CNS use. A good CNS drug should have high specificity and potency, as well as good PK, bioavailability and CNS penetration, and finally should be safe. For instance, sildenafil is reported to cross the BBB [S137] and has an IC₅₀ against PDE5 of 6.0 nM and an in vivo half-life of 0.4 hrs in rodents (˜4 hrs in humans) [S135, S138]. However, the selectivity ratio for PDE1, which is expressed in myocardium and blood vessels besides the brain and may result in mild vasodilatatory effects is 180, and that for PDE6, which is expressed only in retina and can transiently disturb vision is equal to 12 [S106, S107]. Evidence for vardenafil ability to cross the BBB is indirect [150], and even if its IC₅₀ against PDE5 is 0.17 nM, the selectivity ratio for PDE6 is equal to 3.5 [S151, S152]. Without being bound by theory, tadalafil, cannot cross the BBB. Thus, our laboratories have launched a program to develop new PDE5 inhibitors based on knowledge of structures of existing PDE5 inhibitors with a) high specificity and potency, b) great PK properties and CNS penetration, and c) safety, to be used in AD.

Many PDE5 inhibitors have been developed in the past decades and numerous potent compounds have been reported in the literature. Hence, we avoided wasting resources to develop an entirely new scaffold with high potency and excellent selectivity. Instead we focused on known inhibitors and performed a SAR analysis of the existing scaffolds to choose a structure that can lead to the discovery of a class of compounds that can be helpful in the treatment of AD. Rather than choosing cGMP-based molecules such as sildenafil and vardenafil, or β-carbolines-derived molecules such as tadalafil, we identified quinoline derivatives as the top candidates for the design and synthesis of PDE5 inhibitors to be optimized against AD, based on the high potency and selectivity of BMS4. This compound contains important features of two other potent inhibitors, BMS2 and E1 (FIG. 45). It was reported as the most potent and selective PDE5 inhibitor ever identified to date [S19]. Although the in vitro tests of this compound reached our criteria for potency and selectivity for PDE1-6, its selectivity for the remaining PDEs, in vivo efficacy in an AD model or other diseases, PK including BBB penetration, toxicity, and solubility remain unknown. In addition, only a few substituents on the quinoline ring were investigated and just one compound was dominant. Thus, we synthesized YF012403 (FIG. 24), to verify the potency and selectivity of this scaffold, as well as its effectiveness against synaptic and cognitive loss by Aβ, and also to explore the possibility of modifying the scaffold for lead optimization in view of developing a drug that can effectively counteract synaptic and memory loss in AD.

Starting from 2-bromo-4-cyanoaniline, YF012403 was prepared in six steps after reduction of the ester which was obtained by cross-coupling of cyclopropyl boronic acid with quinoline bromide 7 in the presence of Pd(PPh₃)₄. The coupling precursor 7 can be synthesized using the procedure described in [S19]. Of note, the organometal catalyzed cross-coupling reaction leaves us with a great freedom for further modification of this scaffold. For example, by using Buchwald-Hartwig reaction conditions, the 8-dimethyl analog of YFO12403, YF016203, was synthesized. In vitro assays showed that these two compounds have great inhibitory activity against PDE5 and selectivity against all other PDE isoforms (see FIG. 27). The IC₅₀'s of YF012403 and YF016203 for PDE5 are 0.27 nM (FIG. 47) and 0.4 nM, respectively. Neither one of these compounds inhibits any of the other PDEs (PDE5/PDE>1000).

We then investigated the PK and BBB penetration capability of YF012403. After p.o. administration at 50 mg/kg to BABL/c mice, plasma and brain concentrations were determined by the LC-MS/MS. The plasma and brain concentrations at each sampling time are shown in FIG. 48. The data in FIG. 28 indicate that YF012403 is rapidly absorbed as illustrated by the peak plasma concentration occurring at 0.5 h after dosing. Moreover, the T_(max) values in the brain and plasma were similar, indicating that the distribution of YF012403 to the brain is also fast. Finally, the amount of YF012403 in the brain was lower than that in the plasma with an AUC_(0-t) ratio of 0.41 and the elimination half-lives of YF012403 in the brain and plasma were 1.04 and 1.33, respectively.

Our next goal was to check if YF012403 can attenuate synaptic and cognitive dysfunction in APP/PS1 mice. We induced LTP or contextual fear memory in the presence of oligomeric Aβ₄₂, or vehicle [S18]. In the LTP experiments 200 nM Aβ₄₂ or vehicle were perfused through the bath solution for 20 min prior to the application of the θ-burst. In the behavioral experiments 200 nM Aβ₄₂ or vehicle were bilaterally infused 15 min prior to the foot shock into dorsal hippocampus of the animal that had been pre-implanted with a cannula the week before. Aβ reduced LTP and contextual fear memory (FIG. 49). However, YF012403 (50 nM, for 10 min prior to the O-burst in the LTP experiments; 3 or 10 mg/Kg, p.o., immediately after training in the behavioral experiments) ameliorated the electrophysiological and behavioral deficits (FIG. 49). Taken together, these results indicate that YF012403 is a good compound for optimization.

Computational modeling is reliable for predicting the pde inhibitor activity and selectivity. Although X-ray crystal structures of PDE1 to PDE5, PDE7, and PDE9 [S27-S35] have been reported, the fine structure at the active site, for example, whether OH⁻ (hydroxide anion), or a H₂O (water molecule) is the 2nd bridging ligand (BL2) is uncertain because hydrogen atoms can not be determined by X-ray diffraction technique. Using various state-of-the-art computational techniques: molecular dynamics (MD) [S21], first-principles quantum mechanics (QM) [S22], in-house developed first-principles QM-fully polarizable continuum model (QM/FPCM) method [S21, S2, S36-S50] and hybrid QM/molecular mechanics (QM/MM) [S21], HO⁻ (hydroxide anion), not H₂O [142-154] was discovered to be the BL2 in the reported PDE crystal structures. Since the hydroxide anion (HO⁻) is expected to serve as the nucleophile that initiates the catalytic hydrolysis of the substrate [S21, S22], these findings provide a base to construct an accurate 3D structural model, which is critical for performing homology modeling for each of the other PDE families whose X-ray crystal structures are unknown. These newly determined fine 3D structures of PDE5 and other PDEs provide a unique opportunity to help finding selective PDE5 inhibitors that work for CNS.

Medicinal chemistry strategy to design and synthesize new PDE5 inhibitors which are optimized for AD. Our data show that enhancement of the NO/sGC/cGMP/PKG/CREB pathway through inhibition of PDE5 counteracts Aβ-induced synaptic and cognitive abnormalities. Drugs that both act on the NO/sGC/cGMP/PKG/CREB pathway and are optimized for the CNS are lacking. We will obtain new drugs with a) high specificity and potency, b) good PK, bioavailability and CNS penetration, and c) safety. None of the existing drugs is known to fit all of these criteria. Thus, Computer-Aided MedChem Strategy is being used to develop compounds that fit the criteria described herein.

Functional assays will be used in testing PDE5 modulating compounds, such as PDE5 inhibitors, in addition to the parallel use of a validated in vivo disease model. Compounds will be selected according to the following criteria: high potency, excellent selectivity, a reasonable PK profile, and good BBB penetration. Based on the availability of high resolution X-ray structures of PDE5 complexed with sildenafil, vardenafil and tadalafil, in silico calculations will be used to determine druggability and permeability of the designed structures. The compounds with the highest scores will be synthesized. Compounds with satisfactory potency and selectivity will be studied further for PK, bioavailability/brain penetration, and other safety profiles.

Our research design will focus on modifications of YF012403 to optimize its druggability. YF012403 has a primary benzylic alcohol at the 3-position (C3), which can be oxidized by microsomes generating benzaldehyde and consequently causing first-pass metabolism problems and severe side effects due to subsequent conjugate addition to proteins. Its half-life may therefore also be dramatically limited. Thus, it is necessary to convert the benyzlic alcohol into other more drug-friendly groups. Secondly, YF012403 bears a cyclopropyl group at the 8-position (C8) which may not be stable in vivo by undergoing ring opening, and thus representing an electrophilic liability. To avoid this problem, we will change the cyclopropyl to other substituents. Thirdly, the logBB of YF012403 is only −0.38, and therefore is not ideal for drugs against CNS diseases (a peak brain/blood concentration ratio>1 is comparable with that of known CNS drugs in clinical use). Finally, because some 3-cyanoquinoline derivatives have shown inhibitory activity against the NF-κB and other kinases (see for instance [S51-S56]) and replacing the 3-cyano group with an ester group or an alcohol has been found to eliminate these activities entirely [S53, S55], we will modify the group at C3 to minimize the off-target activity. Therefore, we describe structures focusing on modification of the moieties at both the C3 and the C8 positions. In addition, we will modify other parts of the quinoline to improve the pharmacological properties of the top candidates to avoid other ADMET problems.

Modifications at C8. Previous work has indicated that the modifications at C8 are very critical for the inhibitory activity of the new compound [S19]. Although only hydrogen and an ethyl group at the C8 position of the quinoline were explored, given that the ethyl group gave the best result, a bulkier group at C8 would yield better activity. This allows us to pursue an even larger variation at this position, such as positioning cycloalkyl, heterocyclic groups, or alkylamino groups at the C8 position, in order to identify the best substituents at this position. Similar to the preparation of YF012403, these compounds can be synthesized by coupling reactions aided by organometallic catalysis in the presence of Pd, Cu or Fe, such as Heck coupling, Negishi coupling, and Buchwald-Hartwig coupling reactions, starting from the halides which are accessible using the reported procedure [S19](FIG. 50).

Modifications at C3. The reduction of the C3-ethyl ester to the corresponding alcohol lowers the IC₅₀ by one order of magnitude and increases the selectivity over PDE6 by 70-fold. It is unclear whether or not the improvement of activity is due to the fact that an electron-withdrawing group has been replaced by an electron-donating group on the aryl ring, or the hydrogen bond between the resulting alcohol and the 5-amine, or the necessity of an H-acceptor/donor supplied by the free hydroxyl group. However, as noted above, the benzylic alcohol and cyano group at the C3 position can cause potential problems. Thus, several strategies described herein can help to optimize the structure at the C3 position.

The ester/ether L02, thioether/thioester L03, amine/amide L04 can be easily obtained by reaction of the benzyl chloride or benzyl mesylate derived from the free alcohol L01 with alcohol/acid, thiol/thiolacid, or amine/amide in the presence of base, respectively. The above-mentioned benzyl chloride/mesylate can also be substituted by a heterocycle to afford L05. Reaction of alcohol L01 or thiol (L03, R′═H) or amine (L04, R′═H) with triphosgene/carbonyldiimidazole (CDI) or thionyl chloride will lead to the cyclic urea/carbamide (L06), and cyclic thiourea/sulfonamide (L07), respectively. These simple conversions will allow us to rapidly construct several compound libraries derived from the benzylic alcohol that will be able to improve the druggability of this scaffold. In addition, since these compounds may have lower tPSAs, they may have better BBB permeability than the polar alcohol (FIG. 51).

In another strategy, fluorine can be introduced at C3 because usually fluorinated compounds have good PK and an intra-molecular F—H bond can increase the lipophilicity, BBB penetration and bioavailability. Reaction of alcohol L01 with Deoxo-fluor® affords the benzyl fluoride L08. Conversion of L01 to its corresponding aldehyde followed by reaction with Deoxo-fluor® gives the difluoro derivative L09. The trifluoromethyl analog L10 is readily obtained by reaction of Deoxo-fluor® with 3-carboxylic quinoline (FIG. 52).

Introduction of an amino group at the C3 position can be realized via a Curtis rearrangement from the azide derived from 3-carboxylic quinoline. With the 3-amino-quinoline L11 in hand, the 3-fluoro derivative can be prepared, employing Sandmeyer reaction conditions in the presence of HBF₄. Treatment of L11 with an alkyl halide, 1,2-dibromoethane, triphosgene/carbonyldiimidazole (CDI) or thionyl chloride yields other derivatives L13-L16 (FIG. 53).

Modifications at other parts. As our studies proceed, our SAR database will be expanded further and the best substituents at C3 and C8 will be identified. To further improve the pharmacological properties and druggability of this scaffold, we can also modify other parts of this scaffold. Starting from the substituted aniline L17, 3-hydroxy quinoline L19 can be prepared by treatment of L17 with methylenemalonate followed by cyclization at elevated temperatures. Refluxing L19 with POCl₃ would yield 3-chloroquinoline L20, which can then be converted to 3-amino derivative L21 by treatment with ammonia. Treatment of L19 and L21 with different electrophiles would give amide/sulfonamide L22 and ester/sulfonate/ether L24, respectively. Both L22 and L24 will then be converted to the desired derivatives (L23 and L25) based on the SAR studies at C3 described above in section b (FIG. 54).

The structures described herein cover numerous variations and some of them may not be good PDE5 inhibitors with improved selectivity, BBB permeability, PK, and/or other pharmacological properties. Thus, to avoid wasting limited resources before the actual synthesis is begun computational chemistry will be used to assist in prioritizing and identifying top-tier candidates based on docking and AMDET parameter (such as clogP, tPSA, clogBB) calculations. In addition to that, during the optimization process, attention must be given to the previously obtained data so that the subsequent investigations can be guided accordingly.

Computational Strategy to Design and Synthesize New PDE5 Inhibitors which are Optimized for AD.

PDE5 inhibitory properties will be optimized using a computational design consisting of several major stages. Stage 1 involves initial structure-based virtual screening (with a rigid enzyme structure) through de novo design or combinatorial library docking. The computational methods used in the Stage 1 are very fast and, therefore, useful for an automated screening of a large number of virtual molecules or molecular fragments. The highly ranked virtual compounds from Stage 1 will be further considered in Stage 2 for more sophisticated flexible docking. A limited number of virtual compounds (the top-100 or less) that pass Stage 2 will undergo more sophisticated MD simulations of microscopic binding of PDE5 in water and MM-PBSA binding free energy calculations (Stage #3). Next, the selectivity of the predicted PDE5 inhibitors will be evaluated in Stage 4. Among the structures described herein, those being shown by these computational studies will be synthesized and tested for enzymatic activity, so that we will employ a fully integrated approach including medicinal chemistry, computational studies and analysis of drug activity.

i. Stage #1. Initial structure-based virtual screening: Two computational approaches will be used to perform automated large-scale virtual screening with a rigid enzyme structure: de novo design and combinatorial library docking. Both approaches, to be used in an automated way, have their own advantages and thus complement each other. The de novo ligand design is based on a detailed analysis of microscopic enzyme-ligand binding and considers the binding site of a known enzyme or receptor. The structure analysis of existing PDE5 inhibitors and our recently determined 3D structure of the binding site allow us to determine a class of pharmacophore/scaffold with high potency. This pharmacophore/scaffold can then be used as the basis for de novo design of ligands for the receptor. Given the 3D structure of the enzyme, one may identify the subsites of interaction that would ideally be fulfilled by a ligand. A computer program then compares fragments from a database to the interaction subsites, with hits proposed according to scoring rules that reflect real binding. This automated comparison predicts favorable combinations of fragments in different subsites of interaction. A number of computer programs can provide fragment combination methods, including LUDI [S183-S185], CLIX [S186], SPLICE [S187], GroupBuild [S188], and SPROUT [S189-S191]. We have chosen the LUDI program for this project. The program will be able to position molecular fragments into the interaction subsites in such a way that favorable interactions can be formed with the enzyme. Each combination of fragments will then be connected into a single virtual molecule whose ability to bind PDE5 will be scored.

In the combinatorial library docking approach, we will construct a virtual combinatorial library with a docking strategy (using the DOCK6.0 program [S192]), and then the docking of each of these virtual compounds with the PDE5 active site. A commonly used docking strategy is to dissect a ligand into a scaffold and rigid sub-structure fragments, and then to generate new molecular structures by probing many different fragments in a combinatorial fashion. After removing the original fragments (substituents) from the lead compound, we will screen the “fragment”-like compounds in the ZINC database [S193] by docking these fragments in multiple positions and orientations into the subsites of the PDE5 active site. The top fragments (e.g. 500) for each subsite will be ranked and used by the CombilibMaker™ program (Tripos, Inc.) to build a combinatorial library composed of (500)^(n) virtual compounds. When only two subsites are considered for each round of computational design, we will have n=2 and (500)^(n =)250,000 compounds built from each lead compound. Finally, each virtual compound in the combinatorial library will be docked into the PDE5 active site and its binding scored by the automated flexible docking function of the DOCK6.0 program [S192]. Based on the relative values of the docking scores determined by using the DOCK6.0 program and also the docking geometries, the top-scored compounds will be selected for further evaluation in the next stage. These approaches allow us to investigate the possibility of further modification of the quinoline scaffold.

ii. Stage #2. Flexible docking and BBB penetration prediction: The binding structures predicted in Stage #1 for the top scored compounds with a rigid enzyme structure will be further refined and rescored by using the Amber score approach implemented in the DOCK6.0 program [S192]. During the Amber scoring calculation, the input coordinates and parameters of the enzyme-ligand complex will be read into the system. Then, energy minimization using the conjugate gradient method will be performed to optimize the enzyme-ligand contacts. The energy minimization will be followed by a (short-time) Langevin MD simulation at constant temperature and, finally, a short energy-minimization to obtain the final energetic results of the system. Compounds that have both the best docking scores and reasonable docking geometries will be selected for further evaluation in the next stage.

We will also theoretically estimate the BBB penetration by calculating the polar surface area (PSA) and the oil/water partition coefficient (logP) of each candidate compound and using the well-established quantitative structure-activity relationship (QSAR) and artificial neural network (ANN) models [S194, S195]. These QSAR and ANN models have demonstrated that the BBB penetration of a compound is determined by the PSA and logP (or PSA and molecular weight) of the compound, both of which can be calculated conveniently by commercially available software. Usually, compounds that can cross the BBB should have a molecular mass less than 450 Da and a PSA smaller than 90.2 Å² [S194].

iii. Stage #3. MD simulation in water and MM-PBSA binding free energy calculation: For each of top scored compounds that pass both the flexible docking and BBB penetration tests in Stage #2, we will further perform MD simulation on the PDE5-ligand complex. The MD simulation will be performed in a water bath using Amber program suite [S196] with the new-generation force field developed by Cornell et al [S197]. NAMD program (using the same Amber force field or CHARMM force field) [S198] will also be used for massively parallel MD simulations [S199-S207]. Finally, the stable trajectories of MD simulations will be used to perform more sophisticated molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) [S208, S209] binding free energy calculations; the detailed MM-PBSA protocol to be used in this project and its high accuracy in predicting protein-ligand binding have been described and discussed in detail in our recently accomplished computational studies of the inhibitions of PDEs and other proteins [S204, S210]. The calculated binding free energy is a theoretical indicator of the binding affinity for a ligand binding with the enzyme (PDE5). Only compounds predicted to have higher binding affinity for PDE5 will be evaluated in Stage #4 for their selectivity.

v. Stage #4. Computational evaluation of the selectivity of the predicted PDE5 inhibitors: Based on the modeled 3D structures of all PDE families, a new PDE5 inhibitor with an improved selectivity may be designed in such a way that the inhibitor not only keeps the good interactions with the common residues of PDEs, but also has improved interactions with non-common residues (e.g. F787, L804, I813, and M816) of PDE5. Although these non-common residues are not unique for PDE5, no other PDE family has all of these non-common residues. A PDE5 inhibitor is expected to be selective, if it has favorable interactions with all of these non-common residues in addition to the common residues. We will model the binding of the compounds with a virtual library and with other PDEs to assess their selectivity. The results will indicate that some compounds in the virtual library are expected to have a significantly lower binding affinity with other PDEs.

We will use the 3D models of the PDE structures and repeat the flexible docking in Stage #2 (and, if necessary, MD and MM-PBSA calculations in Stage #3) for each of the predicted PDE5 inhibitors binding with other PDE families. The predicted new compounds that are potentially potent and selective for PDE5 (i.e. the predicted IC₅₀<50 nM and the predicted selectivity >100-fold) will be submitted for chemical synthesis and biochemical assays. The actual outcome of the wet experimental tests will be used to refine the computational design protocol and improve the rational basis for subsequent predictions.

Compound analysis. All compounds synthesized and tested for biological activity will be fully characterized and purified to >95% as determined by HPLC and ¹H NMR. Furthermore, additional analytical techniques (i.e. ¹³C NMR, IR, melting point, MS and/or elemental analysis) will be used to determine structure and purify. In the case of optically pure materials, the purity will be assessed by chiral stationary-phase HPLC. In certain cases where structural uncertainty remains other techniques (i.e. 2-D NMR, and x-ray crystallography) will be utilized.

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Example 8 Expression Levels of PDE5 mRNA in Heart, Whole Brain, Hippocampus and Cerebrum of Humans

Quantitative RT-PCR was performed using SYBR green and three different set of primers. Primers specificity was confirmed with a melting curve. Total RNA was prepared by Clontech Laboratories, Inc. The target of Primer-1,2 and 3 was the 3′UTR of PDE5 mRNA.

Primer-1 forward: 5′-TGATGCAAAGCAGGTGAAACC-3′, (SEQ ID NO: 3) Reverse: 5′-ATCCAAGGCCATTCCATTTCT-3′, (SEQ ID NO: 4) Primer-2 forward: 5′-TTCCATGTGCTAGCCAGGTAAA′, (SEQ ID NO: 5) Reverse: 5′-GGTCCAAAACCATGCACAATTT-3′, (SEQ ID NO: 6) Primer-3 forward: 5′-ACCGTGCCAATCACAATCCT′-3′, (SEQ ID NO: 7) Reverse: 5′-AGCTGCCTTCTGTGACATTCTG-3′. (SEQ ID NO: 8)

We have demonstrated that there are very high levels of PDE5 messenger in human hippocampus, even higher than in heart (see FIG. 42). This observation is consistent with database of human brain Gene Logic's ASCENTA System. To perform these experiments was important for our project in view of previous attempts from other groups that cannot demonstrate the presence of messenger in human hippocampi because of two reasons: a) rodent sequence was used to detect human RNA; b) given that the PDE5 gene has a long 3′UTR (more than 5000 bp long). We chose the 3′UTR while others used the coding sequence, and selection of the coding sequence involves more than a 6000 bp long cDNA. Therefore, quantification of mRNA expression carried out by other groups was not correct.

Example 9 Effect of Sildenafil in a Different Animal Model for AD

Fragments of APP which are expressed in the APP/PS1 animals other than Aβ can contribute to alterations in memory. Therefore, we also validated our findings on a different AD model by administering sildenafil (3 mg/kg, i/p.) to mutant human APP(V717F, K670M, N671L) mice, also known as J20 mice. We repeated the same experimental protocol as for the APP/PS1 animals. Mice were divided into 4 groups: J20 with sildenafil (n=10), J20 with vehicle (n=7), WT with sildenafil (n=8) and WT with vehicle (n=8). We found no difference in the freezing behavior among the 4 groups during the training phase of the FC. 24 hrs later we found a decreased freezing in vehicle-treated J20 mice compared to vehicle-treated WT littermates in the analysis of the contextual learning (FIG. 55A). However, sildenafil treatment immediately after the training improved contextual learning in the Tg animals. We did not find a difference in freezing behavior during cued learning among the four groups of mice. Sildenafil also improved spatial working memory in the J20 mice that were daily injected immediately after the training for 3 weeks (FIG. 55B). When the same animals were tested with a visible platform task, no sensory-motor impairment was seen among the four groups.

Example 10 Inhibition of PDE5 and Synaptic Function, Memory and Aβ Load in an AD Mouse Model

Memory loss, synaptic dysfunction and accumulation of amyloid β-peptides (Aβ) are major hallmarks of Alzheimer's disease (AD). Down-regulation of the nitric oxide/cGMP/cGMP-dependent-protein kinase/c-AMP Responsive Element Binding Protein (CREB) cascade has been linked to the synaptic deficits following Aβ elevation. Here we report that the phosphodiesterase 5 inhibitor (PDE5) sildenafil (Viagra), a molecule that enhances phosphorylation of the memory molecule CREB through elevation of cGMP levels, is beneficial against the AD phenotype in a mouse model of amyloid deposition. We demonstrate that the inhibitor produces an immediate and long-lasting amelioration of synaptic function, CREB phosphorylation and memory. This effect is also associated with a long-lasting reduction of Aβ levels. Given that side effects of PDE5 inhibitors are largely known and do not preclude their administration to a senile population, these drugs have potential for the treatment of AD and other diseases associated with elevated Aβ levels.

Introduction

Alzheimer's disease (AD) is characterized by neuronal loss, extracellular senile plaques and intracellular neurofibrillary tangles, leading to memory loss. AD begins as a synaptic disorder produced at least in part, by Aβ (Selkoe, 2002). Long-term-potentiation (LTP), a cellular model of memory, and phosphorylation of CREB, a transcription factor involved in memory, are reduced by Aβ (Vitolo et al., 2002). Interestingly, both nitric oxide (NO) donors and cGMP-analogs counteract the Aβ-induced impairment in LTP and CREB phosphorylation (Puzzo et al., 2005). Vice-versa, genetic ablation of NO-synthase 2 (NOS2) results in worsening of the AD phenotype in mice expressing mutated amyloid precursor protein (APP) (Colton et al., 2006), indicating that up-regulation of the NO pathway may be protective in AD.

One effective way to up-regulate the NO pathway is by increasing cGMP levels through inhibitors of phosphodiesterase 5 (PDE5), an enzyme expressed in several brain regions associated with cognitive function, such as the hippocampus, cortex and cerebellum (Van Staveren et al., 2003; van Staveren et al., 2004) (see also for human brain Gene Logic's ASCENTA System and personal communication from M. Sakurai). Preclinical studies have shown that the selective PDE5 inhibitors sildenafil (Viagra by Pfizer) and vardenafil (Levitra by Bayer) raise hippocampal cGMP levels and improve memory in aged rats (Prickaerts et al., 2002a) and mice (Baratti and Boccia, 1999). Interestingly, FDA has recently approved the daily use of the inhibitor tadalafil (Clalis by Lilly) (see http://www.clinicaspace.com/news story.aspx?NewsEntityld=82124). Moreover, PDE5 inhibitors are widely used to treat erectile dysfunction and pulmonary hypertension, so that their side effects are known. Without being bound by theory, PDE5 inhibitors can be compatible with administration to a senile population such as AD patients. Based on these elements, in the present study we have investigated whether PDE5 inhibition can exert beneficial effects against the AD phenotype of mice carrying both the mutant amyloid precursor protein (APP; K670N,M671L) and presenilin-1 (PS1; M146L), termed APP/PS1 mice.

Materials and Methods

Animals. Double transgenic mice expressing both the human APP (K670M:N671L) and PS1 (M146L) (line 6.2) mutations were used and handled as described in EXAMPLE 1.

Drug preparation. Drug preparation was carried out as described in EXAMPLE 1.

Drug administration. Three-month-old APP/PS1 and WT mice were separated into 4 groups: APP/PS1 mice treated with vehicle, APP/PS1 mice treated with PDE inhibitor, WT mice treated with vehicle, and WT mice treated with PDE inhibitor. In one experimental series we assessed the acute effects of PDE inhibition on synaptic dysfunction by perfusing hippocampal slices with sildenafil (50 nM), or tadalafil (50 nM) or IC354 (1 μM) for 10 min prior to the theta burst. In a separate series of experiments we also examined the acute effect of different concentrations of sildenafil to establish its minimal effective concentration. In the remaining experiments we i.p. injected sildenafil. For assessment of the short-term effects of sildenafil, the drug was given at a concentration of 3 mg/kg immediately after the training. This dose yields concentrations of ˜2.5 μM cGMP in the hippocampus (Prickaerts et al., 2002b). In these experiments, we also established the minimal effective concentration of sildenafil and the minimal effective days of sildenafil delivery. For assessment of long-term effects, sildenafil was given daily by i.p. injection at a concentration of 3 mg/kg for 3 weeks and then treatment was stopped for 9-12 weeks prior to behavioral testing. The minimal effective number of days of sildenafil delivery and the minimal sildenafil effective concentration that can still trigger long-term rescue of memory at 6 months were also studied. Contextual and cued fear conditioning was performed for 3 days. Radial-arm water-maze (RAWM) was performed for 3 weeks. Morris water maze lasted 3 days. Then, the animals were sacrificed for electrophysiological recordings.

To decide the time of administration of sildenafil in the short-term effect experiments, we performed a series of preliminary studies in which the inhibitor was injected i.p. at 5 min before the electric shock or at 5 min before the first acquisition trial with the RAWM. We found no beneficial effect both on the freezing time and the number of errors in sildenafil-injected APP/PS1 mice (sildenafil-treated APP/PS1 mice demonstrated a freezing time equal to ˜90% that of vehicle-treated APP/PS1 mice; n=7 males for sildenafil-treated transgenics and 6 males for vehicle-treated transgenics, P>0.05; ˜5 errors in the retention trial for both sildenafil- and vehicle-treated transgenics, n=6 males for sildenafil-treated transgenics and 5 males for vehicle-treated transgenics, P>0.05, sildenafil did not affect the behavioral performance of WT mice in both tasks, n=5 males for all the conditions). Thus, all the behavioral experiments on the short-term effects of sildenafil reported in the result section were performed with injection after the training. Finally, in a separate set of experiments, we tested the effect of i.p. injection of tadalafil on memory.

Electrophysiological analysis. Electrophysiological analyis was carried out as described in EXAMPLE 1.

Patch clamp experiments were also perfomed to assess both NMDA and AMPA receptor currents. The technique has been previously described (Puzzo et al., 2008). Briefly, 350 μm hippocampal slices were cut with a vibratome and maintained in a submerged chamber at 29° C., perfused with artificial cerebrospinal fluid containing (in mM): 125 NaCl, 2.5 KCl, 1.25 Na₂HPO₄, 25 NaHCO₃, 2 CaCl₂, 1.4 MgCl₂, 25 glucose, 0.1 picrotoxin, pH 7.4 (95% O₂, 5% CO₂). Slices were permitted to recover for 30 min at 37° C. and then at least for 60 min at room temperature before recordings. Neurons were voltage clamped throughout the experiment. Patch pipettes (4-6 MΩ) contained a solution (in mM): 117.5 Cs-methyl-sulfonate, 17.5 CsCl2, 4 NaCl, 0.1 EGTA, 10 HEPES, 5 QX-314.Cl, 4 MgATP, 0.3 Na₂GTP, 10 phosphocreatine-Tris, pH adjusted to 7.3 with CsOH, osmolarity adjusted to 290 mOsm with sucrose. Currents were recorded with a Warner amplifier (PC-501A) and filtered at 1 kHz (holding potential, −70 mV). The amplitude was measured automatically by using the Clampfit program (version 10.1) from Molecular Devices. The AMPAR/NMDAR receptor ratio was calculated by dividing the amplitude of the AMPAR current measured at the peak response at −70 mV by the NMDAR current measured 30 ms after the peak at −50 mV.

Behavioral Studies

Fear conditioning—Studies were carried out as described in EXAMPLE 1.

Spatial working memory—Studies were carried out as described in EXAMPLE 1.

Reference memory—Studies were carried out as described in EXAMPLE 1.

Visible platform testing—Training was carried out as described in EXAMPLE 1.

Immunocytochemical experiments. Immunocytochemical experiments and measurements were carried out as described in EXAMPLE 1.

Determination of Aβ levels. Frozen hemi-brains were weighed and homogenized in 5 M guanidine HCL/50 mM Tris HCL solution. Aβ₄₀ and Aβ₄₂ were measured using human β amyloid ELISA kits (Biosource, CA), according to the manufacturer's protocol. ELISA signals were reported as the mean±s.e.m. in nanograms of Aβ per milligram of cortex. Alternatively, Aβ levels can be determined on homogenates of frozen hemi-brains as previously described [Trinchese et al., Ann Neurol, 2004. 55 (6): p. 801-14]. Aβ can be trapped with either monoclonal antibody to Aβ₄₀ (JRF/cA 40/10) or Aβ₄₂ (JRF/cA 42/26) and then be detected with horseradish peroxidase-conjugated JRF/A tot/17 [Janus et al., Nature, 2000. 408 (6815): p. 979-82]. ELISA signals can be reported as the mean of two replica wells in fmol Aβ per mg protein (determined with the BCA Protein Assay Reagent Kit, PIERCE), based on standard curves using synthetic Aβ₄₀ and Aβ₄₂ peptide standards (American Peptide). Blood can be harvested in a tube containing 10 mM EDTA, then centrifuged at 4000 rpm for 5 min at 40° C. Plasma can then be stored at −80° C. before loading onto ELISA plates.

Statistical analyses. Statistical analysis was carried out as described in EXAMPLE 1. Nonlinear regression analysis was used to fit curves for different concentrations using GraphPad Prism software (GraphPad Software Inc., San Diego, Calif.).

Results

Acute effects of sildenafil on synaptic function in hippocampal slices of APP/PS1 mice. Our first goal was to determine whether PDE5 inhibition can ameliorate synaptic function. Because sildenafil is reported to cross the blood brain barrier (BBB) (FDA, 1998), whereas evidence for vardenafil is indirect (Prickaerts et al., 2004), and tadalafil does not cross it, we chose this compound as a primary drug inhibiting PDE5 for our CNS studies. Sildenafil has an IC₅₀ against PDE5 of 6.0 nM and an in vivo half-life of 0.4 hrs in rodents (˜4 hrs in humans) (Walker et al., 1999; Daugan et al., 2003b). We first tested whether a brief application of sildenafil rescued the defect in LTP of slices derived from 3 month-old APP/PS1 mice, when synaptic plasticity impairment is just starting whereas basal synaptic transmission (BST) is normal (Trinchese et al., 2004). BST was determined by plotting the peak amplitude of the fiber volley against the slope of the field excitatory postsynaptic potential (fEPSPs) and also the fEPSP slope at increasing stimulus intensity in APP/PS1 and wild-type (WT) mice. We did not find a difference in BST among different groups (FIG. 56A, FIG. 67). Hippocampal slices were then perfused with sildenafil (50 nM) for 10 min before inducing LTP through tetanic stimulation of the Schaeffer collateral pathway. Potentiation in sildenafil treated APP/PS1 slices was far greater than in vehicle-treated APP/PS1 slices (FIG. 1B). On the other hand, sildenafil did not change the amplitude of LTP in slices of WT mice compared to WT slices treated with vehicle alone (FIG. 1C). Sildenafil had no effect on basal synaptic responses either during its application or 120 minutes after the end of the application in experiments where no tetanus was applied either in slices from APP/PS1 mice or WT littermates (FIG. 1B,C). Use of different concentrations of sildenafil showed that 50 nM was the minimum dose of the drug that completely rescued synaptic plasticity in slices from transgenic animals, whereas lower concentrations were less effective (FIG. 56B). The same concentrations of the inhibitor did not have an effect on LTP of WT slices. In additional experiments, 50 nM sildenafil ameliorated LTP in slices from APP/PS1 mice that were potentiated through 1 or 2 series of theta-burst stimulations (FIG. 56C). Interestingly, as previously shown on slices from WT mice that received a weaker tetanic stimulation paired with agonists of the NO pathway (Puzzo et al., 2005), 50 nM sildenafil increased the LTP amplitude in WT slices that received 1 theta-burst stimulation (FIG. 56C).

As a control for PDE5 specificity of the sildenafil effect onto synaptic dysfunction, we next used a more specific PDE5 inhibitor, tadalafil. Differently than sildenafil and vardenafil which are cGMP based inhibitors, tadalafil is a (3-carbolines-derived drug with no effect on PDE1 (selectivity ratio>2000) and on PDE6 (selectivity ratio 1000), and an IC₅₀ against PDE5 of 5.0 nM (Daugan et al., 2003b). When slices were bathed in tadalafil (50 nM, 10 min prior to tetanus), potentiation in APP/PS1 slices was far greater than in vehicle-treated APP/PS1 slices (FIG. 8A). Tadalafil did not affect baseline and LTP in WT mice (FIG. 8B).

As an additional control for PDE5 specificity, we have also used a highly selective PDE1 inhibitor called IC354, the HCl salt of IC224 [IC₅₀ against PDE1 of 80 nM; ratio of IC₅₀ value for the next most sensitive PDE to IC₅₀ value for PDE1 equal to 127 (Snyder et al., 2005)]. Differently than sildenafil or tadalafil, when APP/PS1 slices were bathed in IC354 (1 μM, 10 min prior to tetanus), LTP was not affected (FIG. 8C). IC354 did not change LTP amplitude in hippocampal slices of WT mice (FIG. 8D). Thus, these results taken together with the experiments with sildenafil and tadalafil demonstrate that inhibition of PDE5 (but not PDE 1) protects AD-like animal models against synaptic dysfunction, supporting that inhibition of PDE5 can be beneficial against synaptic dysfunction in AD.

Acute effects of sildenafil on the cognitive function of APP/PS1 mice. As reported above, sildenafil offers the advantage of crossing the BBB and therefore it can be easily utilized in behavioural experiments. We divided 3 month-old mice into 4 groups: APP/PS1 with sildenafil, APP/PS1 with vehicle, WT with sildenafil and WT with vehicle. Sildenafil and vehicle control solutions were administered i.p. at a concentration of 3 mg/kg. This concentration was chosen based on previous studies showing that these amounts of sildenafil raise hippocampal cGMP levels and improve memory in aged rats (Prickaerts et al., 2002a) and mice (Baratti and Boccia, 1999) independent of vascular effects (Prickaerts et al., 2002a). We first examined the effects of acute administration of sildenafil on fear-conditioning learning, a type of learning that is impaired in several AD mouse models (Gong et al., 2004b), and depends on hippocampus and amygdala (Phillips and LeDoux, 1992). For contextual fear conditioning, mice were trained to associate neutral stimuli with an aversive one. They were placed in a novel context (fear conditioning box), exposed to a white noise cue (conditioned stimulus, CS) paired with a mild foot shock (unconditioned stimulus, US), and injected with sildenafil immediately after the training. Fear learning was assessed twenty-four hours later by measuring freezing behaviour—the absence of all movement except for that necessitated by breathing—in response to representation of the context or of the auditory cue within a completely different context. We found no difference in the freezing behaviour among the four groups of mice before the training phase (FIG. 2A). Twenty-four hours later, we found a decrease in the freezing behaviour of vehicle-treated APP/PS1 mice compared with that of vehicle-treated WT littermates in the analysis of the contextual learning (FIG. 2A). Sildenafil treatment improved contextual learning in the transgenic animals (FIG. 2A) whereas sildenafil-treated WT animals did not show a significant increase in freezing (FIG. 2A), probably because maximal levels of memory are already induced in vehicle-treated WT mice after the training session, as has been found both in Drosophila and in mice (Tully et al., 2003; Gong et al., 2004a). We next tested cued fear conditioning, a hippocampus-independent task (Phillips and LeDoux, 1992), and did not find a difference in freezing among the 4 groups (FIG. 59), as APP/PS1 mice are known to have a selective hippocampus-dependent impairment in associative learning (Gong et al., 2004b). Moreover, as for the electrophysiological experiments, we determined the minimum concentration of sildenafil needed to improve contextual fear memory in APP/PS1 mice by injecting 1.5 mg/kg, 3 mg/kg and 6 mg/kg inhibitor. A concentration of 3 mg/kg fully restored fear memory (FIG. 58A). No memory enhancement was observed in WT littermates injected with the different concentrations of inhibitor.

Next, we examined the effect of treatment with sildenafil on spatial working memory, a type of short-term memory that can be studied with the RAWM test. This task has already demonstrated memory deficits in other transgenic models of AD (Morgan et al., 2000; Trinchese et al., 2004) and has been shown to depend upon hippocampal function (Diamond et al., 1999). Mice were required to learn and memorize the location of a hidden platform in one of the arms of a maze with respect to spatial cues. APP/PS1 injected with vehicle showed severe abnormalities in spatial memory for platform location during both acquisition and retention of the task compared to vehicle-injected WT littermates (FIG. 2B). However, daily injections of sildenafil for 3 weeks immediately after the 4^(th) acquisition trial ameliorated the behavioural performance of APP/PS1 mice (FIG. 2B). Treatment with sildenafil did not affect the performance of WT mice compared to vehicle-injected WT littermates (FIG. 2B). We also determined the minimum concentration of sildenafil needed to improve spatial working memory in APP/PS1 mice by injecting the drug for 3 weeks with 1.5 mg/kg, 3 mg/kg and 6 mg/kg inhibitor. A concentration of 3 mg/kg fully restored memory (FIG. 58B). Then, we tested the minimum time needed for sildenafil to have a positive effect on spatial working memory. Daily injections of 3 mg/kg sildenafil improved APP/PS1 mouse performance after 2 weeks (FIG. 58C). The four groups of mice showed no difference in the time needed to find the platform in the visible platform task, as well as in swimming speed (FIG. 10). Thus, vision, motor coordination, or motivation were not affected in the four groups of mice and cannot influence the RAWM results.

An interesting difference between the results with RAWM and fear conditioning was related to the fact that sildenafil produces a partial rescue with the RAWM experiments in APP/PS1 mice, whereas rescue was complete with contextual fear conditioning. To exclude that this difference was due to an incomplete formation of memory in the WT mice facilitating the task of sildenafil to equalize memory between transgenic and WT littermates, we performed an additional series of experiments in which we increased the intensity of the foot shock from 0.50 mA to 0.75 mA. This procedure is known to increase the amount of freezing. Regardless of the amounts of freezing, sildenafil fully restores memory in APP/PS1 mice, unlike the RAWM experiments and like the experiments with lower intensity of the foot shock (FIG. 60).

To exclude the possibility that sildenafil produced its behavioural effect through a peripheral vascular action, we repeated the memory studies using tadalafil which is unable to cross the BBB (cLogP=1.43 and information from its manufacturer). Tadalafil (1 mg/kg, i.p.) did not improve either contextual fear conditioning or spatial working memory in APP/PS1 mice. Thus, the effect of sildenafil cannot be due to inhibition of PDE5 in the vascular compartment (FIG. 61).

Persistent effects of sildenafil on cognitive and synaptic functions in APP/PS1 mice. Previous studies have demonstrated that the PDE4 inhibitor rolipram has a prolonged beneficial effect on synaptic and cognitive abnormalities in APP/PS1 mice that persists beyond the administration of the inhibitor (Gong et al., 2004a). This finding has opened a very interesting therapeutic perspective when using drugs up-regulating CREB phosphorylation in AD: a brief course of treatment can be beneficial for a long time. To check whether the same effect is present following sildenafil treatment, we examined whether the PDE5 inhibitor maintains its protective effect against synaptic dysfunction and memory loss. In these experiments, both APP/PS1 and WT mice of 3 months of age were injected i.p. with 3 mg/kg/day sildenafil for 3 weeks, then the treatment was stopped for 9-12 weeks prior to testing. The mice were next subjected to training for contextual learning. As in the acute experiments, when the animals were reintroduced into the same context in which they had been trained 9-12 weeks before, the freezing time was greatly increased in APP/PS1 mice that had been previously treated with sildenafil compared to vehicle-treated APP/PS1 littermates (FIG. 3A). Sildenafil did not increase the freezing time in WT littermates compared to WT mice treated with vehicle (FIG. 3A). There were no differences between the 4 groups in the cued conditioning test. 3 mg/kg was the minimum dose of inhibitor that produced the prolonged beneficial effect on contextual fear memory (FIG. 62A) and 2 weeks were the minimal effective number of days of sildenafil delivery (FIG. 62B). These data indicate that inhibition of PDE5 protects fear contextual learning in APP/PS1 mice for an extended time beyond the duration of drug administration.

The effects of one course of 3-week treatment with sildenafil on spatial working memory were next tested using the RAWM task. There was a difference between the number of errors made by vehicle-treated APP/PS1 and WT mice (FIG. 3B) (Trinchese et al., 2004). Administration of sildenafil for 3 weeks, 9-12 weeks prior to the testing, reduced the gap between the two groups without affecting performance of the WT animals (FIG. 3B). In addition, consistent with the experiments with fear conditioning, 3 mg/kg was the minimum dose of inhibitor that produced the prolonged beneficial effect on spatial working memory (FIG. 62C) and 2 weeks were the minimal effective number of days of sildenafil delivery (FIG. 62D). These data indicate that one course of long-term treatment with the PDE5 inhibitor protects spatial working memory in APP/PS1 mice.

To investigate sildenafil effect on long-term memory, we tested reference memory with a Morris water maze task that is known to require hippocampal function (Schenk and Morris, 1985) and is impaired after 6 months of age in the APP/PS1 mice (Trinchese et al., 2004). Vehicle-treated transgenic mice needed more time to find the hidden platform after six sessions compared to WT littermates (FIG. 3C). When APP/PS1 mice were previously treated with sildenafil they showed a marked improvement of their behavioural performance. Sildenafil did not affect the performance in WT littermates (FIG. 3C). We also assessed reference memory with the probe trial, another test of spatial reference memory (Schenk and Morris, 1985). After the sixth hidden-platform session the platform was removed from the water and the animals were allowed to search for 60 seconds. The mouse is thus indicating that it knows the position of the platform independently of such tactile cues as hitting the platform. Vehicle-treated WT mice spent more time in the target quadrant (TQ), where the platform had been located during training, than in the other quadrants (FIG. 3D). In addition, sildenafil improved the performance of the APP/PS1 mice which searched in the TQ more than the vehicle-treated APP/PS1 mice (FIG. 3D). Sildenafil-treated WT mice remembered where the platform was the previous days and spent about the same time as vehicle-treated WT littermates. Furthermore, consistent with the experiments with fear conditioning and RAWM, 3 mg/kg was the minimum dose of inhibitor that produced the prolonged beneficial effect on reference memory (FIG. 62E,F) and 2 weeks were the minimal effective number of days of sildenafil delivery (FIG. 62G,H). A visible platform trial performed after the probe trials did not reveal any difference in the time to reach the platform and swimming speed among the 4 groups (FIG. 11).

To add depth to the analysis of the functional changes that underlie the striking effects of sildenafil on APP/PS1 mice behavioral performance, we examined synaptic function in hippocampi from the same mice. In contrast to 3-month-old double transgenic mice, 8- to 9-month old APP/PS1 animals show a reduction of synaptic strength (Trinchese et al., 2004). Previous treatment with sildenafil in APP/PS1 mice produced greater values of fEPSP slope in slices from 8 to 9 month old mice than in vehicle-treated APP/PS1 slices (FIG. 4A). On the other hand, sildenafil did not change responses in WT littermates. CA3-CA1 connections that had been tested for BST were also assessed for their capacity of undergoing potentiation. LTP values recorded from slices obtained from APP/PS1 that had been previously treated with sildenafil were similar to their sildenafil treated-WT littermates and far greater than those from vehicle-treated APP/PS1 littermates (FIG. 4B, C). Eight-to nine-month old WT mice showed similar amounts of potentiation whether treated with sildenafil or with vehicle (FIG. 4C). No differences were noted in the baseline transmission of the four groups of mice in the absence of tetanus (FIG. 4B, C). 3 mg/kg was the minimum dose of inhibitor that produced the prolonged beneficial effect on BST and LTP (FIG. 63A,B) and 2 weeks were the minimal effective number of days of sildenafil delivery for rescuing these phenomena (FIG. 63C,D). These data indicate that one course of sildenafil treatment protects APP/PS1 mice against synaptic dysfunction for a long time.

In an additional experimental series, we also examined whether treatment of hippocampal slices from 6 month-old APP/PS1 mice with sildenafil produces an immediate improvement of synaptic function. In contrast to BST which was not ameliorated by the compound, LTP reached normal levels with 500 nM sildenafil (FIG. 64A,B). Thus, once the damage of synaptic function is established, PDE5 inhibition can quickly counteract defects in synaptic plasticity, but not deficits in basal synaptic function.

Effects of sildenafil on creb phosphorylation and all levels in APP/PS1 mice. Given that the duration of action of sildenafil is relatively short, a direct effect of the PDE5 inhibitor cannot be held responsible for its long-term effects. CREB has been implicated in the regulation of genes whose expression results in the formation and stabilization of long-term memory. CREB phosphorylation is required for CREB ability to bind to CREB binding protein (CBP) and to stimulate CRE dependent gene expression (Silva et al., 1998). Aβ elevation is also known to block the tetanus-induced increase in phosphorylation of the memory molecule CREB (Puzzo et al., 2005; Gong et al., 2006). Thus, we measured levels of CREB phosphorylation in sildenafil- and vehicle-treated transgenic and WT mice. Hippocampal slices were treated as described in the electrophysiological experiments, fixed 60 minutes after the treatment, stained with anti-phospho-CREB antibodies at Ser-133, and viewed on a confocal microscope. We confirmed previous findings (Lu et al., 1999; Puzzo et al., 2005) showing an increase in immunofluorescence intensity in the CA1 cell body area of WT mice after tetanus compared to control non-tetanized slices (FIG. 5A,B). APP/PS1 animals did not have the physiological increase of CA1 phospho-CREB immunofluorescence after tetanus (FIG. 5A,B). However, sildenafil re-established normal phospho-CREB increase in tetanized slices of the double transgenics (FIG. 5A,B). Sildenafil did not affect the tetanus-induced increase in immunofluorescence in WT animals (FIG. 5A,B).

We obtained similar results when we investigated mice that had been injected with 3 mg/kg/day sildenafil or vehicle at the age of 3 months and then left without treatment for 9-12 weeks. Similar to the younger animals we found an increase in immunofluorescence intensity in CA1 cell body area of WT mice after tetanus compared to non-tetanized control slices (FIG. 5C). APP/PS1 mice did not reveal the physiological increase of phospho-CREB after tetanus but previous treatment with sildenafil re-established it (FIG. 5C). Moreover, phospho-CREB immunofluorescence did not vary in slices from sildenafil-treated WT mice with tetanic stimulation (FIG. 5C). Thus, at the root of the long-term improvement in synaptic physiology and behaviour there is the re-establishment of the increase of CREB phosphorylation in APP/PS1 mice following tetanic stimulation of the Schaffer collateral-CA1 connection.

What does it underlie the long-lasting improvement in CREB phosphorylation in the APP/PS1 mice? To address this question, given that Aβ down-regulates phospho-CREB, we examined whether sildenafil affects Aβ levels. ELISA of extracts of cerebral cortices revealed a reduction in human Aβ₄₀ and Aβ₄₂ levels in sildenafil-treated APP/PS1 mice after 3 week treatment with 3 mg/kg and 6 mg/kg sildenafil at 3 months and 7-10 months (FIG. 65A,B). Treatment with 1.5 mg/kg, in turn, did not decrease Aβ levels. Finally, when we determined the Aβ levels in animals treated with 3 mg/kg sildenafil for different durations, Aβ levels were already reduced after 2 weeks both in animals that were sacrificed immediately after the treatment (FIG. 65C) and animals that had been injected with 3 mg/kg/day sildenafil at the age of 3 months and then left without treatment for 9-12 weeks (FIG. 65D). Thus, a reduction in AP3 levels is the basis of the prolonged beneficial effect by sildenafil on phospho-CREB.

Discussion

The present study demonstrates that a treatment with the PDE5 inhibitor sildenafil rescues synaptic and memory deficits in a transgenic mouse model of amyloid deposition. Sildenafil also re-establishes the increase in phosphorylation of the transcription factor and memory molecule CREB. In addition, the inhibitor counteracts the negative effects of high levels of Aβ on synaptic function, memory and CREB phosphorylation not only immediately, but also for a prolonged period beyond the drug administration. Finally, sildenafil causes an immediate and long-lasting reduction in Aβ₄₀ and Aβ₄₂ levels. These findings support a model in which PDE5 inhibitors counteract the deficit in CREB phosphorylation by Aβ not only immediately, but also for a prolonged period of time through regulation of transcription of genes controlling Aβ synthesis/degradation.

A relevant finding of the present study is the reversal of the memory impairment in the APP/PS1 mouse following PDE5 inhibition. These results are in agreement with the observation that NO-mimetic molecules may reverse the cognitive impairment caused by scopolamine (Thatcher et al., 2004), or by forebrain cholinergic depletion (Bennett et al., 2007), indicating that stimulating the NO/cGMP signal transduction system can provide new, effective treatments for cognitive disorders. With regard to the beneficial effect on memory, it is interesting to note that inhibition of PDE5 activity during a narrow time window immediately after training for fear learning or after acquisition of the spatial task (but not 5 min before training for fear learning or acquisition of the spatial task) improves learning in the transgenic animals. Considering that the in vivo half-life of sildenafil is 0.4 hrs in rodents (Walker et al., 1999), there is a time-window during the first 20-25 min after the electric shock or the acquisition of the spatial task during which learning processes are susceptible of improvement by PDE5 inhibition. Moreover, given that the beneficial effect of sildenafil was observed with its injection after the training, inhibition of PDE5 acts on memory consolidation mechanisms, and not on aspects of performance, such as perception of pain or of the environment.

Another discovery reported in our study is the beneficial effect of sildenafil against synaptic dysfunction in the APP/PS1 mouse. This finding is consistent with studies on slices showing that cGMP increase through the use of NO donors or cGMP analogs rescues the reduction of LTP and the inhibition of CREB phosphorylation induced by exogenous application of Aβ (Puzzo et al., 2005). Given that altered synaptic function is a fundamental aspect in the cognitive decline of AD (Masliah, 1995), an advantage of using PDE5 inhibitors in AD can be that this class of compounds will counteract aspects of the disease linked to synaptic dysfunction that can be relevant to memory loss.

Decrease in Aβ levels by PDE5 inhibition in transgenic mice is another important finding of our studies. This result is in agreement with the observation that the NO-releasing drug NCX-2216 reduces Aβ load in APP/PS1 mice (Jantzen et al., 2002). Moreover, genetic deletion of NOS2 increases Aβ levels in APP overexpressing mice (Colton et al., 2006). Interestingly, the decrease in Aβ levels was still present after 3 to 5 months from the end of sildenafil administration. Considering that sildenafil has a short half-life, this effect can be due to an action on expression of genes regulating Aβ production and/or clearance. CREB has been implicated in the regulation of genes whose expression results in the formation and stabilization of long-term memory probably through the formation of new synaptic connections (Tully et al., 2003). When phospho-CREB binds to CBP, it stimulates CRE dependent gene expression. CBP functions as a co-activator that facilitates interactions with the basal transcription machinery by working as an acetyltransferase that catalyzes acetylation of the histone H3 of the chromatin, causing a loss in chromosomal repression and increase in the transcription of memory associated genes. Histone acetylation can be self-perpetuating, creating a functionally stable chromatin state and thus chronic changes in the rates of specific gene expression (Battaglioli et al., 2002; Lunyak et al., 2002; Turner, 2002). Without being bound by theory, the prolonged beneficial effect of sildenafil is due to a permanent increase in hystone acetylation. Consistent with this, we have recently demonstrated that inhibition of histone de-acetylation that is normally due to a group of enzymes with a reverse effect of CBP, re-establishes normal LTP and memory in APP/PS1 mice (Francis, Y. I., et al. in Soc Neurosci. Abstr. 548.545, San Diego, 2007).

The beneficial effect of sildenafil resembles many aspects of the effects of rolipram, a PDE4 inhibitor that elevates cAMP levels and therefore activates CREB through PKA in experiments in which it was used the same experimental paradigm as in the present studies (Gong et al., 2004b). Moreover, several nonspecific PDE inhibitors, such as caffeine, papaverine and isobutylmethylxanthine have been reported to improve some behavioral performance in experimental animals, probably by antagonizing adenosine receptors or by acting on intracellular Ca²⁺ stores (Villiger and Dunn, 1981; Randt et al., 1982; Nicholson, 1990; Nehlig et al., 1992). Nevertheless, the beneficial effect of sildenafil can be specific to PDE5 inhibition because tadalafil, a highly selective PDE5 inhibitor reproduced the effect of sildenafil on synaptic dysfunction, whereas IC354, a selective inhibitor of PDE 1, another PDE that can be inhibited by sildenafil (selectivity ratio 180) (Daugan et al., 2003b) did not re-establish normal LTP in slices from the double transgenic mice. Moreover, differently than rolipram which did not improve spatial working memory immediately after its administration, sildenafil immediately augmented spatial working memory. Most importantly, a striking difference between the effect of sildenafil and those of rolipram is that the former reduced Aβ levels in the brains of APP/PS1 mice, whereas the latter did not affect Aβ load.

When proposing a new class of drugs as therapeutic agents it is imperative to consider their side effects. This can determine the failure of PDE4 inhibitors to enhance memory. An advantage of using PDE5 inhibitors is that their side effects are known as they have already been utilized for many years, such that FDA has recently authorized the daily use of tadalafil. Priapism has been reported to occurr in a few cases following the intake of PDE5 inhibitors. However, the current view about the cause of priapism is that it is due to a dysregulation of PDE5 function following down-regulation of the NO pathway (Champion et al., 2005), a phenomenon also caused by Aβ increase (Puzzo et al., 2005)—such that, PDE5 inhibitors have been proposed as therapeutic agents against priapism (Burnett et al., 2006; Rajfer et al., 2006). Additional adverse events of the PDE5 inhibitors include mild vasodilatory effects such as headache, flushing, dyspepsia, and nasal congestion or rhinitis, which can warrant caution in proposing PDE5 inhibitors as AD agents. However, although Aβ is primarily accumulating in the CNS, Aβ is also present in the blood of patients affected by AD and other neurological disorders characterized by abnormal Aβ production (Basun et al., 2002; Andreasen et al., 2003). Intriguingly, systemic Aβ potentiates vasoconstriction not only in cerebral vasculature but also in other districts of the vascular system (Pasquier and Leys, 1998; Khalil et al., 2002; Kalaria, 2003; Suhara et al., 2003; Gentile et al., 2004; Price et al., 2004; Smith et al., 2004). Moreover, hypertension is often associated with AD (Pasquier and Leys, 1998; Gentile et al., 2004; Price et al., 2004). Thus, it is very appealing to think that PDE5 inhibitors can counteract not only CNS symptoms, but also vascular symptoms that often affect AD patients.

Our findings strongly support that inhibition of PDE5 can be beneficial against cognitive loss in AD. However, none of the existing commercially available inhibitors, including sildenafil, are optimized for the CNS. A good CNS drug should have high specificity and potency, as well as good pharmacokinetic, bioavailability and CNS penetration, and finally should be safe. For instance, sildenafil is reported to cross the BBB (FDA, 1998) and has an IC₅₀ against PDE5 of 6.0 nM and an in vivo half-life of 0.4 hrs in rodents (−4 hrs in humans) (Walker et al., 1999; Daugan et al., 2003b). However, the selectivity ratio for PDE1, which is expressed in myocardium and blood vessels besides the brain and may result in mild vasodilatatory effects is 180, and that for PDE6, which is expressed only in retina and can transiently disturb vision is equal to 12 (Corbin and Francis, 2002; Daugan et al., 2003a). Evidence for Vardenafil ability to cross the BBB is indirect (Prickaerts et al., 2004), and even if its IC50 against PDE5 is 0.17 nM, the selectivity ratio for PDE6 is equal to 3.5 (Saenz de Tejada et al., 2001; Zhang et al., 2005). Without being bound by theory, tadalafil, cannot cross the BBB. Thus, our findings support developing new PDE5 inhibitors that are optimized for the CNS that can be used in AD patients.

Supplemental Discussion

AMPA- and NMDA-receptor currents were not altered in 3 month old double transgenic mice. Consistent with these findings basal synaptic transmission was normal in APP/PS1 mice of similar age. A careful analysis of the data published in the literature indicates that AMPA receptors are not affected at the earliest stages of the disease. For instance, Chang et al failed to see an impairment of AMPA receptor currents and basal synaptic transmission in 7-8 month old 2×KI mice, whereas at this age LTP was already impaired (Chang et al., 2006). Consistent with these findings, the concentration of Aβ₄₂ that interfered with AMPA receptor function was very high (2 μM) (Hsieh et al., 2006). Moreover, miniature EPSC amplitude was not altered in neurons overexpressing APP in organotypic hippocampal slice cultures (Kamenetz et al., 2003). Similar considerations can be applied to NMDA receptors. The concentration of Aβ was high (1 μM) in a manuscript demonstrating the involvement of NMDA receptors in AD (Snyder et al., 2005). In addition, extracellular Aβ was applied for a prolonged time in order to see an effect on NMDA receptors (Shankar et al., 2007). Thus, AMPA- and NMDA-receptors are not affected at the earliest stages of AD pathology. Rather, our data indicate that LTP intrinsic mechanisms are affected prior to AMPA and NMDA receptor involvement in the disease.

Drugs acting on the NO-cascade have vascular effects that can affect the cognitive performance. Thus, an alternative explanation for the beneficial effect of sildenafil is that the inhibitor works through a vascular effect instead of an intra-neuronal effect. This is unlikely as inhibition of PDE5 re-established normal LTP in slices directly exposed to PDE5 inhibitors. Moreover, although cAMP analogues have been shown to induce more dilatation of cerebral arterioles in the parietal cortex than cGMP analogues (Paterno et al., 1996), only 8-Br-cGMP (but not 8-Br-cAMP) improved memory performance in rodents (Prickaerts et al., 2002) indicating that vascular mechanisms can not account for the cGMP effects. Most importantly, tadalafil that does not cross the BBB did not reproduce the behavioral effects of sildenafil.

Our findings are in agreement with reports showing that upregulation of the NO cascade has a protective effect on Aβ-induced damage in the CNS (McCarty, 1998; Troy et al., 2000; Wirtz-Brugger and Giovanni, 2000). For instance, studies performed on PC12 cells, sympathetic neurons and hippocampal neurons, have shown that treatment with the NO generator S-nitroso penicillamine has a neuroprotective action through nitrosylation that inhibits the pro-apoptotic factor caspase-2 (Troy et al., 2000). Aβ has been found to impair NO generation by different mechanisms including a decrease in NMDA receptor signal transduction (McCarty, 1998), subtraction of NADPH availability to NOS (Venturini et al., 2002), and inhibition of the phosphorylation of the serine-threonine kinase Akt (Suhara et al., 2003). The superior temporal cortex of AD patients shows a reduction in soluble guanylyl cyclase activity (Bonkale et al., 1995). Soluble guanylyl cyclase is decreased following Aβ exposure in brain astroglial cells (Baltrons et al., 2002). PDE activity increase has been found on both isolated blood cells and cultured microglia, in which PDE5 inhibition re-establishes normal vasoactivity and blocks inflammatory response caused by Aβ (Paris et al., 1999). However, NO has also been viewed as a major agent of neuropathology and cell death when it is produced in high quantity. High amounts of NO lead to generation of significant quantity of peroxinitrites that are responsible for oxidative and nitrosative stress in Aβ-induced cell death (McCann, 1997; Tran et al., 2001; Wong et al., 2001; Haas et al., 2002; Xie et al., 2002; Monsonego et al., 2003; Wang et al., 2004). These opposite findings can be reconciled with our findings with the observation that release of low amounts of NO by the constitutive forms of NOS including both the neuronal and the endothelial isoforms, n-NOS and e-NOS, promotes synaptic plasticity and learning, whereas uncontrolled production of high amounts of the gas by the inducible form of NO-synthase (iNOS) may promote oxidative and nitrosative stress via production of peroxinitrite (McCann, 1997; Tran et al., 2001; Wong et al., 2001; Haas et al., 2002; Xie et al., 2002; Monsonego et al., 2003; Wang et al., 2004). The current status of drug research exploiting these discoveries is focused both on finding ways to upregulate the NO cascade and therefore elicit neuroprotection, as well as on finding ways to block peroxinitrite toxic effects in order to limit neuropathology (Contestabile et al., 2003). Our therapeutic strategy intervening with PDE5 offers the advantage of bypassing NO production by focusing on steps at the downstream level of NO generation.

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What is claimed is:
 1. A method for treating a subject afflicted with Alzheimer's disease, Lewy body dementia, inclusion body myositis, or cerebral amyloid angiopathy, the method comprising: administering to the subject an effective amount of a composition comprising a PDE5 inhibitor compound of Formula (V):

wherein: A is O or N; X is —(CH₂)_(n), C(O), S(O), or S(O)₂; R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷; R² is —CH₂OR⁶ or —CO₂R⁸; R³ is hydrogen or halogen; R⁴ is —CN or halogen; R⁵ is hydrogen or —OR⁶; R⁶ is hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹; R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, —NR⁹R¹⁰, —SR⁹, or heterocyclyl; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8-membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with a heteroatom, and wherein the heterocycle is optionally substituted with C₁-C₆ alkyl; and R⁹ and R¹⁰ are each independently hydrogen, C₁-C₆ alkyl, or C₃-C₈ cycloalkyl; and n is 1, 2, or 3, or a pharmaceutically acceptable salt or tautomer thereof.
 2. The method of claim 1, wherein the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, or at least about 10 mg/kg body weight.
 3. The method of claim 1, wherein A is N.
 4. The method of claim 1, wherein R⁵ is hydrogen.
 5. The method of claim 1, wherein R⁵ is —OCH₃.
 6. The method of claim 1, wherein the compound is of formula (V-1):

wherein: R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷; R² is —CH₂OR⁶ or —CO₂R⁸; R³ is hydrogen or halogen; R⁴ is —CN or halogen; R⁶ is hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹; R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, —NR⁹R¹⁰, —SR⁹, or heterocyclyl; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8-membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with a heteroatom, and wherein the heterocycle is optionally substituted with C₁-C₆ alkyl; and R⁹ and R¹⁰ are each independently hydrogen, C₁-C₆ alkyl, or C₃-C₈ cycloalkyl, or a pharmaceutically acceptable salt or tautomer thereof.
 7. The method of claim 5, wherein R⁶ is CH₃.
 8. The method of claim 1, wherein the compound is of formula (V-1a):

wherein: R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷; R² is —CH₂OR⁶ or —CO₂R⁸; R³ is hydrogen or halogen; R⁴ is —CN or halogen; R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, —NR⁹R¹⁰, —SR⁹, or heterocyclyl; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8-membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with a heteroatom, and wherein the heterocycle is optionally substituted with C₁-C₆ alkyl; and R⁹ and R¹⁰ are each independently hydrogen, C₁-C₆ alkyl, or C₃-C₈ cycloalkyl, or a pharmaceutically acceptable salt or tautomer thereof.
 9. The method of claim 1, wherein R² is CH₂—OH.
 10. The method of claim 1, wherein R³ is H.
 11. The method of claim 1, wherein R³ is a halogen.
 12. The method of claim 1, wherein R³ is chlorine.
 13. The method of claim 1, wherein R⁴ is —CN.
 14. The method of claim 1, wherein R⁴ is a halogen.
 15. The method of claim 1, wherein R⁴ is fluorine.
 16. The method of claim 1, wherein the compound is of formula (V-1a1):

wherein: R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷; R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, —NR⁹R¹⁰, —SR⁹, or heterocyclyl; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8- membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with a heteroatom, and wherein the heterocycle is optionally substituted with C₁-C₆ alkyl; and R⁹ and R¹⁰ are each independently hydrogen, C₁-C₆ alkyl, or C₃-C₈ cycloalkyl, or a pharmaceutically acceptable salt or tautomer thereof.
 17. The method of claim 1, wherein R¹ is C₃-C₈ cycloalkyl or —NR⁷R⁸.
 18. The method of claim 1, wherein R¹ is —NR⁷R⁸.
 19. The method of claim 1, wherein R¹ is —NR⁷R⁸, and wherein R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl,or —NR⁹R¹⁰; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8- membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with O, NR⁹ or N—C(O)R⁹.
 20. The method of claim 1, wherein R¹ is —SR₇.
 21. The method of claim 1, wherein R¹ is —S—(C₁-C₆)-alkyl.
 22. The method of claim 1, wherein R¹ is C₃-C₈ cycloalkyl.
 23. The method of claim 1, wherein R¹ is cyclopropyl.
 24. The method of claim 1, wherein R¹ is dimethylamino.
 25. The method of claim 1, wherein the compound is


26. The method of claim 1, wherein the compound is


27. A method for the treatment of at least one condition selected from the group consisting of erectile dysfunction, pulmonary hypertension, cardiovascular disorder, and gastrointestinal disorder in a subject in need of such treatment, the method comprising: administering to the subject an effective amount of a composition comprising a PDE5 inhibitor compound of Formula (V):

wherein: A is O or N; X is —(CH₂)_(n), C(O), S(O), or S(O)₂; R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷; R² is —CH₂OR⁶ or —CO₂R⁸; R³ is hydrogen or halogen; R⁴ is —CN or halogen; R⁵ is hydrogen or —OR⁶; R⁶ is hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹; R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, —NR⁹R¹⁰, —SR⁹, or heterocyclyl; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8-membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with a heteroatom, and wherein the heterocycle is optionally substituted with C₁-C₆ alkyl; and R⁹ and R¹⁰ are each independently hydrogen, C₁-C₆ alkyl, or C₃-C₈ cycloalkyl; and n is 1, 2, or 3, or a pharmaceutically acceptable salt or tautomer thereof.
 28. The method of claim 27, wherein the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, or at least about 10 mg/kg body weight.
 29. The method of claim 27, wherein A is N.
 30. The method of claim 27, wherein R⁵ is hydrogen.
 31. The method of claim 27, wherein R⁵ is —OCH₃.
 32. The method of claim 27, wherein the compound is of formula (V-1):

wherein: R^(i) is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR'; R² is —CH₂OR⁶ or —CO₂R⁸; R³ is hydrogen or halogen; R⁴ is —CN or halogen; R⁶ is hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹; R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, —NR⁹R¹⁰, —SR⁹, or heterocyclyl; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8-membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with a heteroatom, and wherein the heterocycle is optionally substituted with C₁-C₆ alkyl; and R⁹ and R¹⁰ are each independently hydrogen, C₁-C₆ alkyl, or C₃-C₈ cycloalkyl, or a pharmaceutically acceptable salt or tautomer thereof.
 33. The method of claim 32, wherein R⁶ is CH₃.
 34. The method of claim 27, wherein the compound is of formula (V-1a):

wherein: R^(i) is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR'; R² is —CH₂OR⁶ or —CO₂R⁸; R³ is hydrogen or halogen; R⁴ is —CN or halogen; R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, —NR⁹R¹⁰, —SR⁹, or heterocyclyl; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8-membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with a heteroatom, and wherein the heterocycle is optionally substituted with C₁-C₆ alkyl; and R⁹ and R¹⁰ are each independently hydrogen, C₁-C₆ alkyl, or C₃-C₈ cycloalkyl, or a pharmaceutically acceptable salt or tautomer thereof.
 35. The method of claim 27, wherein R² is CH₂—OH.
 36. The method of claim 27, wherein R³ is H.
 37. The method of claim 27, wherein R³ is a halogen.
 38. The method of claim 27, wherein R³ is chlorine.
 39. The method of claim 27, wherein R⁴ is —CN.
 40. The method of claim 27, wherein R⁴ is a halogen.
 41. The method of claim 27, wherein R⁴ is fluorine.
 42. The method of claim 27, wherein the compound is of formula (V-1a1):

wherein: R¹ is C₃-C₈ cycloalkyl, —NR⁷R⁸, or —SR⁷; R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₁-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, —NR⁹R¹⁰, —SR⁹, or heterocyclyl; or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8-membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with a heteroatom, and wherein the heterocycle is optionally substituted with C₁-C₆ alkyl; and R⁹ and R¹⁰ are each independently hydrogen, C₁-C₆ alkyl, or C₃-C₈ cycloalkyl, or a pharmaceutically acceptable salt or tautomer thereof.
 43. The method of claim 27, wherein R¹ is —NR⁷R⁸.
 44. The method of claim 27, wherein R¹ is —NR⁷R⁸, and wherein R⁷ and R⁸ are each independently hydrogen, —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl, or —C(O)R⁹, wherein the C₃-C₆ alkyl or C₃-C₈ cycloalkyl are optionally substituted with —C₁-C₆ alkyl, —C₃-C₈ cycloalkyl,or —NR⁹R¹⁰or, R⁷ and R⁸ together with the nitrogen atom to which they are attached form a 3 to 8- membered heterocycle, wherein any one of the ring carbon atoms is optionally replaced with O, NR⁹ or N—C(O)R⁹.
 45. The method of claim 27, wherein R¹ is —SR⁷.
 46. The method of claim 27, wherein R¹ is —S—(C₁-C₆)-alkyl.
 47. The method of claim 27, wherein R¹ is C₃-C₈ cycloalkyl.
 48. The method of claim 27, wherein R¹ is cyclopropyl.
 49. The method of claim 27, wherein R¹ is dimethylamino.
 50. The method of claim 27, wherein the compound is


51. The method of claim 27, wherein the compound is 